WO2001042509A1 - Use of multiple recombination sites with unique specificity in recombinational cloning - Google Patents
Use of multiple recombination sites with unique specificity in recombinational cloning Download PDFInfo
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- WO2001042509A1 WO2001042509A1 PCT/US2000/033546 US0033546W WO0142509A1 WO 2001042509 A1 WO2001042509 A1 WO 2001042509A1 US 0033546 W US0033546 W US 0033546W WO 0142509 A1 WO0142509 A1 WO 0142509A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
Definitions
- the present invention relates to the fields of biotechnology and molecular biology.
- the present invention relates to joining multiple nucleic acid molecules containing recombination sites, preferably using recombination sites having a unique specificity.
- the present invention also relates to cloning such joined nucleic acid molecules using recombinational cloning methods.
- the invention also relates to joining multiple peptides, and combinations of peptides and nucleic acid molecules through the use of recombination sites.
- Other molecules and compounds or combinations of molecules and compounds may also be joined through recombination sites according to the invention.
- Such peptides, nucleic acids and other molecules and/or compounds (or combinations thereof) may also be joined or bound through recombination to one or a number of supports or structures in accordance with the invention.
- Site-specific recombinases are proteins that are present in many organisms (e g-, viruses and bacteria) and have been characterized as having both endonuclease and ligase properties. These recombinases (along with associated proteins in some cases) recognize specific sequences of bases in a nucleic acid molecule and exchange the nucleic acid segments flanking those sequences. The recombinases and associated proteins are collectively referred to as "recombination proteins" (see, e.g. , Landy, A., Current Opinion in Biotechnology
- Cre// ⁇ xP system frbm bacteriophage PI Hoess and Abremski (1990) In Nucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90- 109
- the FLP/FRT system from the Saccharomyces cerevisiae 2 ⁇ circle plasmid Broach, etal, Cell 29:227-234 (1982)).
- Transposons are mobile genetic elements. Transposons are structurally variable, being described as simple or compound, but typically encode a transposition catalyzing enzyme, termed a transposase, flanked by DNA sequences organized in inverted orientations. For a more thorough discussion of the characteristics of transposons, one may consult Mobile Genetic Elements, D. j. Sherratt, Ed., Oxford University Press (1995) and Mobile DNA, D. E. Berg and M. M. Howe, Eds., American Society for Microbiology (1989), Washington, DC both of which are specifically incorporated herein by reference. Transposons have been used to insert DNA into target DNA. As a general rule, the insertion of transposons into target DNA is a random event.
- Transposon Tn7 can integrate itself into a specific site in the E. coli genome as one part of its life cycle (Stellwagen, A.E., and Craig, N.L. Trends in Biochemical Sciences 23, 486-490, 1998 specifically incorporated herein by reference). This site specific insertion has been used in vivo to manipulate the baculovirus genome (Lucklow et al, J. Virol. 67:4566-4579 (1993) specifically incorporated herein by reference). The site specificity of Tn7 is atypical of transposable elements whose hallmark is movement to random positions in acceptor DNA molecules.
- transposition will be used to refer to random or quasi-random movement, unless otherwise specified, whereas recombination will be used to refer to site specific recombination events.
- site specific insertion of Tn7 into the ⁇ ttTn 7 site would be referred to as a recombination event while the random insertion of Tn7 would be referred to as a transposition event.
- Integrons generally consist of a 5'- and a 3'-conserved sequence flanking a variable sequence. Typically, the 5'-conserved sequence contains the coding information for an integrase protein.
- the integrase protein may catalyze site-specific recombination at a variety of recombination sites including attl, attC as well as other types of sites (see Francia et al, J. Bacteriology 181 (21 ):6844-6849, 1999, and references cited therein).
- recombination sites are sections or segments of nucleic acid on the participating nucleic acid molecules that are recognized and bound by the recombination proteins during the initial stages of integration or recombination.
- the recombination site for Cre recombinase is lox? which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence.
- recognition sequences include the ttB, ⁇ ttP, att , and atlR sequences which are recognized by the recombination protein ⁇ Int.
- ⁇ ttB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region
- ⁇ ttP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis).
- IHF auxiliary proteins integration host factor
- FIS FIS
- Xis excisionase
- Stop Codons and Suppressor tRNAs Three codons are used by both eukaryotes and prokaryotes to signal the end of gene. When transcribed into mRNA, the codons have the following sequences: UAG (amber), UGA (opal) and UAA (ochre). Under most circumstances, the cell does not contain any tRNA molecules that recognize these codons. Thus, when a ribosome translating an mRNA reaches one of these codons, the ribosome stalls and falls of the RNA, terminating translation of the mRNA. The release of the ribosome from the mRNA is mediated by specific factors (see S. Mottagui-Tabar, NAR 26(11), 2789, 1998).
- a gene with an in-frame stop codon (TAA, TAG, or TGA) will ordinarily encode a protein with a native carboxy terminus.
- suppressor tRNAs can result in the insertion of amino acids and continuation of translation past stop codons.
- suppressor tRNAs Mutant tRNA molecules that recognize what are ordinarily stop codons suppress the termination of translation of an mRNA molecule and are termed suppressor tRNAs.
- a number of such suppressor tRNAs have been found. Examples include, but are not limited to, the supE, sup?, supD, supF and supZ suppressors which suppress the termination of translation of the amber stop codon, supB, g ⁇ T, supL, supN, supC and supM suppressors which suppress the function of the ochre stop codon and glyT, trpT and Su-9 which suppress the function of the opal stop codon.
- suppressor tRNAs contain one or more mutations in the anti-codon loop of the tRNA that allows the tRNA to base pair with a codon that ordinarily functions as a stop codon.
- the mutant tRNA is charged with its cognate amino acid residue and the cognate amino acid residue is inserted into the translating polypeptide when the stop codon is encountered.
- the reader may consult Eggertsson, et al, (1988) Microbiological Review 52(3):354-374, and Engleerg-Kukla, et al.
- mutations in the uar gene also known as the r A gene
- mutations in the ups gene mutations in the sueA, sueB and .vweC genes
- mutations in the rpsO (ramA) and rpsE (spcA) genes mutations in the rplL gene.
- the TAA stop codon is the strongest, and most difficult to suppress.
- the TGA is the weakest, and naturally (in E. coli) leaks to the extent of 3%.
- the TAG (amber) codon is relatively tight, with a read-through of ⁇ 1% without suppression.
- the amber codon can be suppressed with efficiencies on the order of 50% with naturally occurring suppressor mutants.
- coli chloramphenicol acetyltransferase (cat) gene having a stop codon in place of the codon for serine 27 was transfected into mammalian cells along with a gene encoding a human serine tRNA which had been mutated to form an amber, ochre, or opal suppressor derivative of the gene. Successful expression of the cat gene was observed.
- An inducible mammalian amber suppressor has been used to suppress a mutation in the replicase gene of polio virus and cell lines expressing the suppressor were successfully used to propagate the mutated virus (Sedivy, et al, (1987) Cell 50: 379-389).
- nucleic acid segments currently occurs as a daily routine in many research labs and as a prerequisite step in many genetic analyses.
- the purpose of these clonings is various, however, two general purposes can be considered: (1) the initial cloning of nucleic acid from large DNA or RNA segments (chromosomes, YACs, PCR fragments, mRNA, etc.), done in a relative handful of known vectors such as pUC, pGem, pBlueScript, and (2) the subcloning of these nucleic acid segments into specialized vectors for functional analysis.
- a great deal of time and effort is expended both in the transfer of nucleic acid segments from the initial cloning vectors to the more specialized vectors. This transfer is called subcloning.
- the basic methods for cloning have been known for many years and have changed little during that time.
- a typical cloning protocol is as follows:
- the specialized vectors used for subcloning nucleic acid segments are functionally diverse. These include but are not limited to: vectors for expressing nucleic acid molecules in various organisms; for regulating nucleic acid molecule expression; for providing tags to aid in protein purification or to allow tracking of proteins in cells; for modifying the cloned nucleic acid segment (e.g., generating deletions); for the synthesis of probes (e.g., riboprobes); for the preparation of templates for nucleic acid sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; to provide large amounts of the nucleic acid of interest, etc.
- nucleic acid segment of interest it is common that a particular investigation will involve subcloning the nucleic acid segment of interest into several different specialized vectors.
- simple subclonings can be done in one day (e.g. , the nucleic acid segment is not large and the restriction sites are compatible with those of the subcloning vector).
- many other subclonings can take several weeks, especially those involving unknown sequences, long fragments, toxic genes, unsuitable placement of restriction sites, high backgrounds, impure enzymes, etc.
- One of the most tedious and time consuming type of subcloning involves the sequential addition of several nucleic acid segments to a vector in order to construct a desired clone.
- This type of cloning is in the construction of gene targeting vectors.
- Gene targeting vectors typically include two nucleic acid segments, each identical to a portion of the target gene, flanking a selectable marker. In order to construct such a vector, it may be necessary to clone each segment sequentially, i.e., first one gene fragment is inserted into the vector, then the selectable marker and then the second fragment of the target gene. This may require a number of digestion, purification, ligation and isolation steps for each fragment cloned. Subcloning nucleic acid fragments is thus often viewed as a chore to be done as few times as possible.
- the system utilizes vectors that contain at least two different site-specific recombination sites based on the bacteriophage lambda system (e.g. , att ⁇ and ⁇ tt2) that are mutated from the wild-type (attO) sites.
- Each mutated site has a unique specificity for its cognate partner all site (i.e., its binding partner recombination site) of the same type (for example ⁇ ttB 1 with ⁇ ttP 1 , or ttL 1 with ⁇ ttRl ) and will not cross-react with recombination sites of the other mutant type or with the wild-type ⁇ ttO site.
- Nucleic acid fragments flanked by recombination sites are cloned and subcloned using the GATEWAYTM system by replacing a selectable marker (for example, ccdB) flanked by att sites on the recipient plasmid molecule, sometimes termed the Destination Vector. Desired clones are then selected by transformation of a ccdB sensitive host strain and positive selection for a marker on the recipient molecule. Similar strategies for negative selection (e.g. , use of toxic genes) can be used in other organisms such as thymidine kinase (TK) in mammals and insects.
- TK thymidine kinase
- each additional mutation potentially creates a novel att site with unique specificity that will recombine only with its cognate partner att site bearing the same mutation and will not cross-react with any other mutant or wild-type ⁇ tt site.
- Novel mutated att sites e.g., attB 1-10, ⁇ ttP 1-10, ⁇ ttR 1-10 and attL 1-10) are described in previous patent application serial number 09/517,466, filed March 2, 2000, which is specifically incorporated herein by reference.
- recombination sites having unique specificity i.e., a first site will recombine with its corresponding site and will not recombine or not substantially recombine with a second site having a different specificity
- suitable recombination sites include, but are not limited to, loxV sites; /oxP site mutants, variants or derivatives such as / ⁇ xP511 (see U.S. Patent No.
- frt sites frt site mutants, variants or derivatives
- the present invention provides novel methods using such recombination sites to join or link multiple nucleic acid molecules or segments and more specifically to clone such multiple segments (e.g.
- the present invention generally provides materials and methods for joining or combining two or more (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, seventy-five, one hundred, two hundred, etc.) segments or molecules of nucleic acid by the recombination reaction between recombination sites, at least one of which is present on each molecule or segment.
- Such recombination reactions to join multiple nucleic acid molecules according to the invention may be conducted in vivo (e.g., within a cell, tissue, organ or organism) or in vitro (e.g. , cell-free systems). Accordingly, the invention relates to methods for creating novel or unique combinations of nucleic acid molecules and to the nucleic acid molecules created by such methods.
- the invention also relates to host and host cells comprising the nucleic acid molecules of the invention.
- the invention also relates to kits for carrying out the methods of the invention, and to compositions for carrying out the methods of the invention as well as compositions made while carrying out the methods of the invention.
- nucleic acid molecules created by the methods of the invention may be used for any purpose known to those skilled in the art.
- the nucleic acid molecules of the invention may be used to express proteins or peptides encoded by the nucleic acid molecules and may be used to create novel fusion proteins by expressing different sequences linked by the methods of the invention. Such expression can be accomplished in a cell or by using well known in vitro expression/transcription systems.
- At least one (and preferably two or more) of the nucleic acid molecules or segments to be joined by the methods of the invention comprise at least two recombination sites, although each molecule may comprise multiple recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.).
- Such recombination sites (which may be the same or different) may be located at various positions in each nucleic acid molecule or segment and the nucleic acid used in the invention may have various sizes and be in different forms including circular, supercoiled, linear, and the like.
- the nucleic acid molecules used in the invention may also comprise one or more vectors or one or more sequences allowing the molecule to function as a vector in a host cell (such as an origin of replication).
- the nucleic acid molecules of the invention may also comprise non-coding segments (e.g., intronic, untranslated, or other segments) that serve a structural or other non-expressive functions.
- the nucleic acid molecules or segments for use in the invention are linear molecules having at least one recombination site at or near at least one termini of the molecule and preferably comprise at least one recombination site at or near both termini of the molecule.
- the corresponding binding partner recombination sites preferably are located on one or more other nucleic acid molecules to be linked or joined by the methods of the invention.
- a first nucleic acid molecule used in the invention may comprise at least a first and second recombination site and a second nucleic acid molecule may comprise at least a third and fourth recombination site, wherein the first and second sites do not recombine with each other and the third and fourth sites do not recombine with each other, although the first and third and/or the second and fourth sites may recombine.
- the nucleic acid molecules to be joined by the methods of the invention are used to produce one or more (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, seventy-five, one hundred, two hundred, etc.) hybrid molecules (e.g., the "product nucleic acid molecules") containing all or a portion of the starting molecules.
- the starting molecules can be any nucleic acid molecule derived from any source or produced by any method.
- Such molecules may be derived from natural sources (such as cells (e.g., prokaryotic cells such as bacterial cells, eukaryotic cells such as fungal cells (e.g., yeast cells), plant cells, animals cells (e.g., mammalian cells such as human cells), etc.), viruses, tissues, organs from any animal or non-animal source, and organisms) or may be non-natural (e.g., derivative nucleic acids) or synthetically derived.
- Such molecules may also include prokaryotic and eukaryotic vectors, plasmids, integration sequences (e.g. , transposons), phage or viral vectors, phagemids, cosmids, and the like.
- the segments or molecules for use in the invention may be produced by any means known to those skilled in the art including, but not limited to, amplification such as by PCR, isolation from natural sources, chemical synthesis, shearing or restriction digest of larger nucleic acid molecules (such as genomic or cDNA), transcription, reverse transcription and the like, and recombination sites may be added to such molecules by any means known to those skilled in the art including ligation of adapters containing recombination sites, attachment with topoisomerases of adapters containing recombination sites, attachment with topoisomerases of adapter primers containing recombination sites, amplification or nucleic acid synthesis using primers containing recombination sites, insertion or integration of nucleic acid molecules (e.g. , transponsons or integration sequences) containing recombination sites etc.
- the nucleic acid molecules used in the invention are populations of molecules such as nucleic acid libraries or cDNA libraries.
- Recombination sites for use in the invention may be any recognition sequence on a nucleic acid molecule which participates in a recombination reaction mediated or catalyzed by one or more recombination proteins.
- recombination sites may be the same or different and may recombine with each other or may not recombine or not substantially recombine with each other.
- Recombination sites contemplated by the invention also include mutants, derivatives or variants of wild-type or naturally occurring recombination sites.
- Preferred recombination site modifications include those that enhance recombination, such enhancements being selected from the group consisting of substantially (i) favoring integrative recombination; (ii) favoring excisive recombination; (iii) relieving the requirement for host factors; (iv) increasing the efficiency of co-integrate or product formation; and (v) increasing the specificity of co-integrate or product formation.
- Preferred modifications to the recombination sites include those that enhance recombination specificity, remove one or more stop codons, and/or avoid hair-pin formation. Desired modifications can also be made to the recombination sites to include desired amino acid changes to the transcription or translation product (e.g., mRNA or protein) when translation or transcription occurs across the modified recombination site.
- Preferred recombination sites used in accordance with the invention include att sites, frt sites, dif sites, psi sites, cer sites, and lox sites or mutants, derivatives and variants thereof (or combinations thereof). Recombination sites contemplated by the invention also include portions of such recombination sites.
- the invention allows directional linking of nucleic acid molecules to provide desired orientations of the linked molecules or non-directional linking to produce random orientations of the linked molecules.
- the recombination sites which recombine with each other in compositions and used in methods of the invention comprise att sites having identical seven base pair overlap regions.
- the first three nucleotides of these seven base pair overlap regions comprise nucleotide sequences selected from the group consisting of AAA, AAC, AAG, AAT, ACA, ACC, ACG, ACT, AGA, AGC, AGG, AGT,
- ATA ATC, ATG; ATT, CAA, CAC, CAG, CAT, CCA, CCC, CCG, CCT, CGA, CGC, CGG, CGT, CTA, CTC, CTG CTT, GAA, GAC, GAG, GAT, GCA, GCC, GCG, GCT, GGA, GGC, GGG, GGT, GTA, GTC, GTG, GTT, TAA, TAC, TAG, TAT, TCA, TCC, TCG, TCT, TGA, TGC, TGG, TGT, TTA, TTC, TTG, and TTT.
- Each starting nucleic acid molecule may comprise, in addition to one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), a variety of sequences (or combinations thereof) including, but not limited to sequences suitable for use as primer sites (e.g., sequences which a primer such as a sequencing primer or amplification primer may hybridize to initiate nucleic acid synthesis, amplification or sequencing), transcription or translation signals or regulatory sequences such as promoters or enhancers, ribosomal binding sites, Kozak sequences, start codons, transcription and/or translation termination signals such as stop codons (which may be optimally suppressed by one or more suppressor tRNA molecules), origins of replication, selectable markers, and genes or portions of genes which may be used to create protein fusion (e.g.
- N-terminal or carboxy terminal such as glutathione S-transferase (GST), ⁇ -glucuronidase (GUS), histidine tags (HIS6), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), open reading frame (ORF) sequences, and any other sequence of interest which may be desired or used in various molecular biology techniques including sequences for use in homologous recombination (e.g., for use in gene targeting).
- GST glutathione S-transferase
- GUS ⁇ -glucuronidase
- HIS6 histidine tags
- GFP green fluorescent protein
- YFP yellow fluorescent protein
- CFP cyan fluorescent protein
- ORF open reading frame
- the invention provides methods for producing populations of hybrid nucleic acid molecules comprising (a) mixing at least a first population of nucleic acid molecules comprising one or more recombination sites with at least one target nucleic acid molecule comprising one or more recombination sites; and (b) causing some or all of the nucleic acid molecules of the at least first population to recombine with all or some of the target nucleic acid molecules, thereby forming the populations of hybrid nucleic acid molecules.
- the recombination is caused by mixing the first population of nucleic acid molecules and the target nucleic acid molecule with one or more recombination proteins under conditions which favor the recombination to produce hybrid nucleic acid molecules.
- methods of the invention further comprise mixing the hybrid nucleic acid molecules with at least a second population of nucleic acid molecules comprising one or more recombination sites to produce a second population of product nucleic acid molecules.
- the first population, second population and target nucleic acid molecules may be mixed together to form a hybrid population through recombination.
- methods of the invention further comprise selecting for the populations of hybrid nucleic acid molecules generated by the methods described above.
- methods of the invention further comprise selecting for the population of hybrid nucleic acid molecules, against the first population of nucleic acid molecules, against the target nucleic acid molecules, and/or against the second population of nucleic acid molecules.
- the invention provides methods for recombining a first nucleic acid segment containing a first recombination site, a second nucleic acid segment containing a second and third recombination site, and a third nucleic acid segment containing a fourth recombination site, wherein the first, second, or third nucleic acid segments may be identical nucleic acid segments or populations of nucleic acid molecules, such that recombination generates a linear or closed, circle product comprising the first, second and third nucleic acid segments.
- members of the recombination products may be amplified using oligonucleotides which either contain or do not contain recombination sites and are homologous or degenerate to the first or third nucleic acid segments.
- oligonucleotides which either contain or do not contain recombination sites and are homologous or degenerate to the first or third nucleic acid segments.
- a product comprising the first-second-third hybrid molecules can be amplified, where other undesired molecules (e.g., products comprising the first-second hybrid molecules) are not amplified.
- amplification can be used to select for desired products and against undesired products.
- Such amplification can be designed to select for any desired products or intermediates of a recombination reaction. For example, four different molecules (e.g.
- A, B, C, and D can be joined and various intermediate products can be selected for (e.g., A-B-C, or A-B) using primers designed to amplify the desired products (e.g., primers corresponding to molecules A and C, when A-B-C is amplified and A and B when A-B is amplified).
- primers designed to amplify the desired products e.g., primers corresponding to molecules A and C, when A-B-C is amplified and A and B when A-B is amplified.
- the resulting amplified products may then be cloned.
- the process described above can be performed using two or more (e.g. , two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fifteen, etc.) nucleic acid segments.
- the invention provides methods of producing populations of hybrid nucleic acid molecules comprising (a) mixing at least a first population of nucleic acid molecules comprising one or more recombination sites with at least a second population of nucleic acid molecules comprising one or more recombination sites; and (b) causing some or all of the nucleic acid molecules of the at least first population to recombine with all or some nucleic acid molecules of the at least second population, thereby forming one or more populations of hybrid nucleic acid molecules.
- recombination is caused by mixing the first population of nucleic acid molecules and the second population of nucleic acid molecules with one or more recombination proteins under conditions which favor their recombination.
- methods of the invention further comprise mixing the first and second populations of nucleic acid molecules with at least a third population of nucleic acid molecules comprising one or more recombination sites. In additional other specific embodiments, methods of the invention further comprise selecting for the population of hybrid nucleic acid molecules. In yet other specific embodiments, methods of the invention further comprise selecting for the population of hybrid nucleic acid molecules and against the first, second, and/or third populations of nucleic acid molecules. In further specific embodiments, methods of the invention further comprise selecting for or against cointegrate molecules and/or byproduct molecules.
- the invention further includes populations of hybrid nucleic acid molecules produced by the above methods and populations of recombinant host cells comprising the above populations of hybrid nucleic acid molecules.
- the recombination proteins used in the practice of the invention comprise one or more proteins selected from the group consisting of Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, TndX, XerC, XerD, and ⁇ C31.
- the recombination sites comprise one or more recombination sites selected from the group consisting of /ox sites; psi sites; dif sites; cer sites; _/rt sites; att sites; and mutants, variants, and derivatives of these recombination sites which retain the ability to undergo recombination.
- the invention allows controlled expression of fusion proteins by suppression of one or more stop codons.
- one or more starting molecules e.g., one, two, three, four, five, seven, ten, twelve, etc.
- joined by the invention may comprise one or more stop codons which may be suppressed to allow expression from a first starting molecule through the next joined starting molecule.
- a first-second-third starting molecule joined by the invention when each of such first and second molecules contains a stop codon) can express a tripartite fusion protein encoded by the joined molecules by suppressing each of the stop codons.
- the invention allows selective or controlled fusion protein expression by varying the suppression of selected stop codons.
- the stop codons may be included anywhere within the starting nucleic acid molecule or within a recombination site contained by the starting molecule. Preferably, such stop codons are located at or near the termini of the starting molecule of interest, although such stop codons may be included internally within the molecule.
- one or more of the starting nucleic acid molecules may comprise the coding sequence of all or a portion of the target gene or open reading frame of interest wherein the coding sequence is followed by a stop codon.
- the stop codon may then be followed by a recombination site allowing joining of a second starting molecule.
- the stop codon may be optionally suppressed by a suppressor tRNA molecule.
- the genes coding for the suppressor tRNA molecule may be provided on the same vector comprising the target gene of interest, on a different vector, or in the chromosome of the host cell into which the vector comprising the coding sequence is inserted.
- more than one copy e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc. copies
- more than one copy e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc. copies
- the transcription of the suppressor tRNA may be under the control of a regulatable (e.g., inducible or repressible) promoter.
- the invention relates to a method of expressing one or more fusion proteins (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) comprising:
- recombination sites described herein may contain stop codons in one, two or all three forward or reverse reading frames. Such termination codons may be suppressed as described above. Further, in appropriate instances, such recombination sites may be designed so as to eliminate stop codons in one, two and/or all three forward and/or reverse reading frames.
- the invention provides methods of synthesizing proteins comprising (a) providing at least a first nucleic acid molecule comprising a coding sequence followed by a stop codon; (b) providing at least a second nucleic acid molecule comprising a coding sequence, optionally, followed by a stop codon; (c) causing recombination such that the nucleic acid molecules are joined; (d) inserting said joined nucleic acid molecules into a vector to produce modified vectors with the two coding sequences connected in frame; (e) transforming host cells which express suppressor tRNAs with the modified vectors; and (f) causing expression of the two coding sequences such that fusion proteins encoded by at least a portion of both of the coding sequences are produced, wherein the nucleic acid molecules of (a) and (b) are each flanked by at least one recombination site.
- the fused nucleic acid molecules or the vector may comprise at least one suppressible stop codon (e.g., amber, opal and/or ochre codons).
- the first or second nucleic acid molecule may already be present in the vector prior to application of the methods described above.
- the vectors and/or host cells comprise genes which encode at least one suppressor tRNA molecule.
- methods of the invention further comprise transforming the host cell with a nucleic acid molecule comprising genes which encode at least one suppressor tRNA molecule.
- the fusion proteins may comprise N- or C-terminal tags (e.g., glutathione S-transferase, ⁇ -glucuronidase, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, maltose binding protein, a six histidine tag, an epitope tag, etc.) encoded by at least a portion of the vector.
- N- or C-terminal tags e.g., glutathione S-transferase, ⁇ -glucuronidase, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, maltose binding protein, a six histidine tag, an epitope tag, etc.
- the invention also relates to a method of expressing one or more fusion proteins (e.g. , one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) comprising: (a) obtaining at least a first nucleic acid molecule comprising at least one recombination site (preferably the recombination site is located at or near a terminus or termini of said first nucleic acid molecule) and a second nucleic acid molecule comprising at least one recombination site (which is preferably located at or near a terminus or termini of said second nucleic acid molecule); (b) causing said at least first and second nucleic acid molecules to recombine through recombination of said recombination sites, thereby producing a third nucleic acid molecule comprising all or a portion of said at least first and second molecules; and
- fusion protein expressing one or more peptides or proteins (e.g. , one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) encoded by said third nucleic acid molecule.
- at least part of the expressed fusion protein will be encoded by the third nucleic acid molecule and at least another part will be encoded by at least part of the first and/or second nucleic acid molecules.
- Such a fusion protein may be produced by translation of nucleic acid which corresponds to recombination sites located between the first and second nucleic acid molecules.
- fusion proteins may be expressed by "reading through" mRNA corresponding to recombination sites used to connect two or more nucleic acid segments.
- the invention further includes fusion proteins produced by methods of the invention and mRNA which encodes such fusion proteins.
- fusion proteins which are encoded by different nucleic acid segments, as well as nucleic acid molecules which encode such fusion proteins.
- the invention provides methods for producing fusion proteins prepared by the expression of nucleic acid molecules generated by connecting two or more nucleic acid segments.
- the invention provides methods for producing fusion RNAs prepared by the expression of nucleic acid molecules generated by connecting two or more nucleic acid segments. These RNAs may be mRNA or may be untranslated RNAs which have activities other than protein coding functions. Examples of such RNAs include ribozymes and tRNAs.
- the invention further provides nucleic acid molecules produced by methods of the invention, expression products of these nucleic acid molecules, methods for producing these expression products, recombinant host cells which contain these nucleic acid molecules, and methods for making these host cells. As discussed below in more detail, the invention further provides combinatorial libraries which may be screened to identify nucleic acid molecules and expression products having particular functions or activities.
- the present invention provides materials and methods for joining two nucleic acid molecules or portions thereof, each of which contains at least one recombination site, into one or more product nucleic acid molecules by incubating the molecules under conditions causing the recombination of a recombination site present on one nucleic acid molecule with a recombination site present on the other nucleic acid molecule.
- the recombination sites are preferably located at or near the ends of the starting nucleic acid molecules.
- the product molecule thus created will contain all or a portion of the first and second starting molecules joined by a recombination site (which is preferably a new recombination site).
- the present invention provides materials and methods for joining two or more nucleic acid molecules (e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) into one or more product nucleic acid molecules (e.g., one, two, three, four, five seven, ten, twelve, etc.) wherein each starting nucleic acid molecule has at least one recombination site and at least one of the starting nucleic acid molecules has at least two recombination sites.
- nucleic acid molecules e.g., two, three, four, five seven, ten, twelve, etc.
- the recombination sites preferably are located at or near one or both termini of the starting nucleic acid molecules.
- the invention provides a method of joining at least two nucleic acid molecules wherein at least a first nucleic acid molecule contains at least one recombination site and at least a second nucleic acid molecule contains two or more recombination sites.
- the molecules are incubated in the presence of at least one recombination protein under conditions sufficient to combine all or a portion of the starting molecules to create one or more product molecules.
- the product molecules thus created will contain all or a portion of each of the starting molecules joined by a recombination site (which is preferably a new recombination site).
- the present invention provides a method to join at least three nucleic acid molecules (e.g. , two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) wherein the molecules have at least one recombination site and at least one of the starting nucleic acid molecules contains at least two recombination sites.
- Incubating such molecules in the presence of at least one recombination protein provides one or more product molecules (e.g., one, two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) containing all or a portion of the starting molecules, wherein each molecule is joined by a recombination site (which is preferably a new recombination site).
- the present invention provides compositions and methods for joining two or more nucleic acid molecules (e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), at least two of which (and preferably all of which) have two or more recombination sites.
- the recombination sites located on each molecule are preferably located at or near the ends of the starting nucleic acid molecules.
- the two or more nucleic acid molecules or portions thereof are joined by a recombination reaction (e.g., incubate the molecules in the presence of at least one recombination protein) to form one or more product molecules comprising all or a portion of each starting molecule joined by a recombination site (which is preferably a new recombination site).
- a recombination reaction e.g., incubate the molecules in the presence of at least one recombination protein
- the present invention provides compositions and methods for joining at least three nucleic acid molecules comprising providing at least a first, a second and a third nucleic acid molecule, wherein the first nucleic acid molecule comprises at least a first recombination site, the second nucleic acid molecule comprises at least a second and a third recombination site and the third nucleic acid molecule comprises at least a fourth recombination site, wherein the first recombination site is capable of recombining with the second recombination site and the third recombination site is capable of recombining with the fourth recombination site and conducting at least one recombination reaction such that the first and the second recombination sites recombine and the third and the fourth recombination sites recombine, thereby combining all or a portion of the molecules to make one or more product molecules.
- the present invention generally relates to a method of combining n nucleic acid molecules or segments, wherein n is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 30, 40, 50, etc.), comprising the steps of providing a 1 st through an n lb nucleic acid molecule or segment, each molecule from 2 through n- ⁇ having at least two recombination sites and molecules 1 and n having at least one recombination site (and preferably having at least two recombination sites), and contacting the molecules or segments with one or more recombination proteins (e.g.
- Joining of molecules through recombination sites preferably creates a new recombination site at the junction of the two molecules and may create a new recombination site at each junction where each molecule is joined to the next.
- the first recombination site on the x molecule interacts with a second recombination site on the y molecule and the second recombination site on the x molecule interacts with a first recombination site on the z molecule to create a hybrid nucleic acid molecule comprising y:x:z joined by recombination sites.
- recombination events may produce hybrid molecules comprising, for example, x:y:z, x:z:y, y:z:x, z:x:y, and/or z:y:x or fragments thereof, joined by recombination sites.
- Additional molecules can be added to product molecules by recombination between at least one recombination site located another molecules with one or more recombination sites located on the product molecule (e.g. , interacting a second recombination site on the z molecule with a first recombination site on an e molecule, etc.
- hybrid nucleic acid molecule comprising y:x:z (or other sequences as noted above) can be circularized by the interaction of recombination sites on the free ends of y and z. Addition of all or a portion of the starting molecules may be done sequentially or simultaneously.
- nucleic acid segments joined by methods of the invention contain a terminus, or termini, which do not contain recombination sites
- this terminus or termini may be connected to the same nucleic acid segment or another nucleic acid molecule using a ligase or a topoisomerase (e.g., a Vaccinia virus topoisomerase; see U.S. Patent No. 5,766,891, the entire disclosure of which is incorporated herein by reference).
- a ligase or a topoisomerase e.g., a Vaccinia virus topoisomerase; see U.S. Patent No. 5,766,891, the entire disclosure of which is incorporated herein by reference.
- the invention also provides a means to replace one or more molecules (or combinations thereof) contained in a product molecule.
- any one or more n molecules comprising the product molecule may be replaced or substituted by recombination with all or a portion of a different molecule (m) which comprises one or more recombination sites.
- m may replace x in the y:x:z molecule described above by recombining a first recombination site on m with the first recombination site flanking x (e.g., the recombination site between y and x) and recombining a second recombination site on m with the second recombination site flanking x
- any nucleic acid molecule of the invention by recombining one or more recombination sites on such molecule with one or more recombination sites within or on the molecule to be substituted.
- one or more deletions e.g., two, three, four, five seven, ten, twelve, etc.
- one or more deletions may be accomplished by recombining two or more recombination sites within the molecule of interest for creating the deletion.
- the invention also provides a means to insert one or more molecules (or combinations thereof) into a product molecule.
- molecule w which comprises one or more recombination sites may be inserted between y and x to form a new molecule: y:w:x:z.
- molecule w is flanked by / ⁇ xP sites and insertion of molecule w is mediated by Cre recombinase between the /oxP sites on the w molecule and corresponding / ⁇ xP sites on the y and x molecules.
- molecule o which comprises one or more recombination sites may be inserted between y and x to form a new molecule comprising either y:o:x:z or y:o:w:x:z, depending on the starting molecule.
- the methods described herein can be used to insert virtually any number of molecules into other molecules. Further, these methods can be used sequentially, for example, to prepare molecules having diverse structures.
- the product molecules produced by the methods of the invention may comprise any combination of starting molecules (or portions thereof) and can be any size and be in any form (e.g., circular, linear, supercoiled, etc.), depending on the starting nucleic acid molecule or segment, the location of the recombination sites on the molecule, and the order of recombination of the sites.
- the present invention provides a means by which populations of nucleic acid molecules (known or unknown) can be combined with one or more known or unknown target sequences of interest (e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or with other populations of nucleic acid molecules (known or unknown), thereby creating populations of combinatorial molecules (e.g. , combinatorial libraries) from which unique and/or novel molecules (e.g., hybrid molecules) and proteins or peptides encoded by these molecules may be obtained and further analyzed.
- target sequences of interest e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- combinatorial molecules e.g., combinatorial libraries
- unique and/or novel molecules e.g., hybrid molecules
- proteins or peptides encoded by these molecules may be obtained and further analyzed.
- the population of nucleic acid molecules used to create combinatorial libraries according to the invention may comprise a population of segments or molecules having at least one (and preferably two or more) recombination sites (e.g., two, three, four, five seven, ten, twelve, etc.).
- populations of molecules are preferably obtained from genomic or cDNA libraries (or portions thereof) or random nucleic acids, amplification products (e.g., PCR products generated with various primers) and domains (e.g., nucleic acids encoding different protein domains from the same or different proteins) constructed to contain such recombination sites.
- a first population of molecules comprising recombination sites can be randomly joined or combined through recombination (by directed and/or random orientation) with at least one target sequence of interest or with a second population of molecules comprising recombination sites to produce a third population of molecules or hybrid molecules.
- multiple populations of molecules from various sources may be combined multiple times to create a new population which comprises molecules having multiple combinations of sequences. For instance, a first population, a second population and a third population can be recombined to create a fourth population comprising a random population of tripartite molecules (e.g., some or all of the molecules of the fourth population contain all or a portion of the segments from the first, second and third population).
- the newly created population of molecules (e.g. , the third population) created by the combinatorial methods may be preferentially selected and thus separated or isolated from the original molecules (e.g., target molecules, and first and second population molecules) and from undesired product molecules (e.g., cointegrates and/or byproduct molecules).
- selection may be accomplished by assaying or selecting for the presence of a desired nucleic acid fusion (PCR with diagnostic primers) and/or the presence of a desired activity of a protein encoded by the desired nucleic acid fusion.
- Such selective may also be accomplished by positive and/or negative selection.
- One or more toxic genes are preferably used according to the invention in such negative selection scheme.
- Combinations of selection of the desired fusion product (nucleic acid and/or protein) and positive and/or negative selection may also be used in the invention.
- the invention provides a means for selecting a population of Product molecules (or even a specific class of product molecules or specific product molecule) created by recombinational cloning and selecting against a population of Insert Donors, Vector Donors and Cointegrates or, in similar fashion, selecting for a population of Insert Donors, Vector Donors, Byproducts and/or Cointegrates and selecting against a population of Product molecules (.see Figure 1).
- a first population of molecules of the invention may be provided as one population of Insert Donor molecules while a second population of molecules, represented by segment B, may be provided as a second population of Insert Donor molecules. While these segments are depicted as linear fragments, they may be provided as segments within a larger molecule, for example, as segments in a plasmid.
- cointegrate molecules other than the one shown in Figure 1 .
- cointegrates comprising a segment A and a segment B Insert Donor molecule may be formed.
- cointegrates comprising segment A and/or segment B Insert Donor molecules and a Vector Donor molecule may be formed.
- the selection methods of the present invention permit selection against the Insert Donor molecules and against the various cointegrate molecules and for the newly created population of hybrid molecules which may be referred to as a population of Product molecules. Conversely, the selection methods may permit selection against Products and for Insert/Vector Donors, Byproducts, and/or Cointegrates.
- the invention relates to a method to create a population of hybrid nucleic acid molecules comprising: (a) mixing at least a first population of nucleic acid molecules comprising one or more recombination sites (e.g. , two, three, four, five seven, ten, twelve, etc.) with at least one target nucleic acid molecule of interest comprising one or more recombination sites (e.g., two, three, four, five seven, ten, twelve, etc.); (b) causing (preferably randomly) some or all of the molecules of said at least first population to recombine with all or some molecules of said target molecule of interest, thereby forming a third population of hybrid molecules; and
- the hybrid molecules contained by the third population preferably comprise all or a portion of a molecule obtained from the first population and all or a portion of the target molecule.
- the orientation in which the molecules are joined may be done in a directed or random manner, depending on the need.
- the target molecule used to produce said third population described above can be a DNA binding domain or a transcription activation domain, such that the third population of hybrid molecules can be used in 2-hybrid screening methods well known in the art.
- the invention more specifically relates to a method of creating a population of combinatorial molecules comprising:
- nucleic acid molecules comprising one or more recombination sites (e.g. , two, three, four, five seven, ten, twelve, etc.) and at least a second population of nucleic acid molecules comprising one or more recombination sites (e.g. , two, three, four, five seven, ten, twelve, etc.);
- each or many of the hybrid molecules contained by the third population preferably comprises all or a portion of a molecule obtained from the first population and all or a portion of a molecule obtained from the second population.
- the orientation which the molecules are joined may be done in a directed or random manner, depending on the need.
- Populations of nucleic acid molecules used in accordance with the combinatorial methods of the invention can comprise synthetic, genomic, or cDNA libraries (or portions thereof), random synthetic sequences or degenerate oligonucleotides, domains and the like.
- the population of nucleic acid molecules used comprises a random population of molecules, each having at least two recombination sites which preferably do not recombine with each other and which are preferably located at or near both termini of each molecule. Random recombination of populations of molecules by the methods of the invention provides a powerful technique for generating populations of molecules having significant sequence diversity. For example, recombination of a first library having about 10 6 sequences with a second population having about 10 6 sequences results in a third population having about 10 12 sequences.
- the invention further provides methods for preparing and screening combinatorial libraries in which segments of the nucleic acid molecules of the library members have been altered. Such alterations include mutation, shuffling, insertion, and/or deletion of nucleic acid segments.
- the invention provides methods for preparing nucleic acid libraries which contain members having such alterations and methods for introducing such alterations in existing libraries.
- the invention includes combinatorial libraries produced by methods of the invention, methods for screening such libraries to identify members which encode expression products having particular functions or activities, and expression products of these libraries (e.g. , RNA, proteins, etc.).
- the invention provides methods for generating populations of nucleic acid molecule containing one or more (e.g., one, two, three, four, five, ten, fifteen) nucleic acid segments which are the same and one or more nucleic acid segments which are derived from members of one or more populations of nucleic acid molecules.
- One method for producing such nucleic acid molecules involves the use of a vector which contains two recombination sites.
- a first nucleic acid segment which encodes a protein having a particular function or activity (e.g., signal peptide activity, DNA binding activity, affinity for a particular ligand, etc.), is inserted in the first recombination site and a second nucleic acid segment, which is derived from a population of nucleic acid molecules, is inserted into the second recombination site. Further, these nucleic acid segments are operably linked to a sequence which regulates transcription, thereby producing a fusion peptide and an RNA molecule produced by the fusion sequence. The resulting combinatorial library may then be screened to identify nucleic acid molecules which encode expression products having particular functions or activities (e.g.
- nucleic acid segments may be kept constant and one or more of the nucleic acid segments may be derived from members of one or more populations of nucleic acid molecules.
- a and D may be known molecules having known functions (e.g. , tags such as HIS6, promoters, transcription or translation signals, selectable markers, etc.) while molecules B and C may be derived from one or more populations of nucleic acid molecules.
- any of the product molecules of the invention may be further manipulated, analyzed or used in any number of standard molecular biology techniques or combinations of such techniques (in vitro or in vivo). These techniques include sequencing, amplification, nucleic acid synthesis, making RNA transcripts (e.g., through transcription of product molecules using RNA promoters such as T7 or SP6 promoters), protein or peptide expression (for example, fusion protein expression, antibody expression, hormone expression etc.), protein-protein interactions (2-hybrid or reverse 2-hybrid analysis), homologous recombination or gene targeting, and combinatorial library analysis and manipulation.
- RNA promoters such as T7 or SP6 promoters
- protein or peptide expression for example, fusion protein expression, antibody expression, hormone expression etc.
- protein-protein interactions (2-hybrid or reverse 2-hybrid analysis
- homologous recombination or gene targeting and combinatorial library analysis and manipulation.
- the invention also relates to cloning the nucleic acid molecules of the invention (preferably by recombination) into one or more vectors (e.g. , two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or converting the nucleic acid molecules of the invention into a vector by the addition of certain functional vector sequences (e.g., origins of replication).
- recombination is accomplished in vitro (e.g., in cell-free systems) and further manipulation or analysis is performed directly in vitro.
- further analysis and manipulation will not be constrained by the ability to introduce the molecules of the invention into a host cell and/or maintained in a host cell.
- less time and higher throughput may be accomplished by further manipulating or analyzing the molecules of the invention directly in vitro.
- in vitro analysis or manipulation can be done after passage through host cells or can be done directly in vivo (e.g., while in the host cells, tissues, organs, or organisms).
- Nucleic acid synthesis steps may comprise: (a) mixing a nucleic acid molecule of interest or template with one or more primers (e.g., one, two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) and one or more nucleotides (e.g., one, two, three, or four) to form a mixture; and (b) incubating said mixture under conditions sufficient to synthesize a nucleic acid molecule complementary to all or a portion of said molecule or template.
- primers e.g., one, two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- nucleotides e.g., one, two, three, or four
- the synthesized molecule may then be used as a template for further synthesis of a nucleic acid molecule complementary to all or a portion of the first synthesized molecule. Accordingly, a double stranded nucleic acid molecule
- nucleic acid molecules e.g., DNA
- synthesis of one or more nucleic acid molecules is performed in the presence of one or more polymerases (preferably DNA polymerases which may be thermostable or mesophilic), although reverse transcriptases may also be used in such synthesis reactions.
- polymerases preferably DNA polymerases which may be thermostable or mesophilic
- nucleic acid molecules used as templates for the synthesis of additional nucleic acid molecules may be RNA, mRNA, DNA or non-natural or derivative nucleic acid molecules.
- Nucleic acid synthesis may be facilitated by incorporating one or more primer sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) into the product molecules through the use of starting nucleic acid molecules containing such primer sites.
- primer sites may be added at one or a number of desired locations in the product molecules, depending on the location of the primer site within the starting molecule and the order of addition of the starting molecule in the product molecule. Sequencing steps, according to the invention, may comprise:
- nucleic acid molecule to be sequenced with one or more primers (e.g., one, two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), one or more nucleotides (e.g., one, two, three, or four) and one or more termination agents (e.g. , one, two, three, four, or five) to form a mixture;
- primers e.g., one, two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- nucleotides e.g., one, two, three, or four
- termination agents e.g. , one, two, three, four, or five
- Such sequencing steps are preferably performed in the presence of one or more polymerases (e.g. , DNA polymerases and/or reverse transcriptases) and one or more primers.
- Preferred terminating agents for sequencing include derivative nucleotides such as dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP and derivatives thereof).
- Nucleic acid sequencing may be facilitated by incorporating one or more sequencing primer sites (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) into the product molecules through the use of starting nucleic acid molecules containing such primer sites.
- sequencing primer sites may be added at one or a number of desired locations in the product molecules, depending on the location of the primer site within the starting molecule and the order of addition of the starting molecule in the product molecule.
- Protein expression steps may comprise:
- nucleic acid molecule to be expressed which comprises one or more expression signals (e.g., one, two, three, or four);
- the expression signal may be said to be operably linked to the sequence to be expressed.
- the protein or peptide expressed can be expressed in a host cell (in vivo), although expression may be conducted in vitro (e.g., in cell-free expression systems) using techniques well known in the art. Upon expression of the protein or peptide, the protein or peptide product may optionally be isolated or purified. Moreover, the expressed protein or peptide may be used in various protein analysis techniques including 2-hybrid interaction, protein functional analysis, and agonist/antagonist-protein interactions (e.g.
- expressed proteins or peptides may be screened to identify those which have particular biological activities. Examples of such activities include binding affinity for nucleic acid molecules (e.g., DNA or RNA) or proteins or peptides. In particular, expressed proteins or peptides may be screened to identify those with binding affinity for other proteins or themselves. Proteins or peptides which have binding affinities for themselves will generally be capable of forming multimers or aggregates.
- Proteins or peptides which have binding affinities for themselves and/or other proteins will often be capable of forming or participating in the formation of multi-protein complexes such as antibodies, splicesomes, multi-subunit enzymes, multi-subunit enzymes, ribosomes, etc. Further included within the scope of the invention are the expressed proteins or peptides described above, nucleic acid molecules which encodes these proteins, methods for making these nucleic acid molecules, methods for producing recombinant host cells which contain these nucleic acid molecules, recombinant host cells produced by these methods, and methods for producing the expressed proteins or peptides.
- hybrid proteins or peptides produced by the invention and particularly from expression of the combinatorial molecules of the invention may generally be useful for any number of applications. More specifically, as one skilled in the art would recognize, hybrid proteins or peptides of the invention may be designed and selected to identify those which to perform virtually any function. Examples of applications for which these proteins may be used include therapeutics, industrial manufacturing (e.g., microbial synthesis of amino acids or carbohydrates), small molecule identification and purification (e.g., by affinity chromatography), etc.
- Protein expression may be facilitated by incorporating one or more transcription or translation signals (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, etc.) or regulatory sequences, start codons, termination signals, splice donor/acceptor sequences (e.g., intronic sequences) and the like into the product molecules through the use of starting nucleic acid molecules containing such sequences.
- transcription or translation signals e.g., one, two, three, four, five, seven, ten, twelve, fifteen, etc.
- regulatory sequences e.g., one, two, three, four, five, seven, ten, twelve, fifteen, etc.
- start codons e.g., start codons
- termination signals e.g., start codons
- splice donor/acceptor sequences e.g., intronic sequences
- the invention provides methods for performing homologous recombination between nucleic acid molecules comprising (a) mixing at least a first nucleic acid molecule which comprises one or more recombination sites with at least one target nucleic acid molecule, wherein the first and target nucleic acid molecules have one or more homologous sequences; and (b) causing the first and target nucleic acid molecules to recombine by homologous recombination.
- the homologous recombination methods of the invention result in transfer of all or a portion of the first nucleic acid molecule into the target nucleic acid molecule.
- the first nucleic acid molecule comprises two or more sequences which are homologous to sequences of the target nucleic acid molecule.
- the homologous sequences of the first nucleic acid molecule flank at least one selectable marker and/or one or more recombination sites.
- the homologous sequences of the first nucleic acid molecule flank at least one selectable marker flanked by recombination sites.
- the homologous sequences of the first nucleic acid molecule flank a nucleic acid segment which regulates transcription.
- homologous recombination may comprise:
- nucleic acid molecule of the invention which is preferably a product molecule
- a first nucleic acid molecule of the invention comprising one or more recombination sites (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.)
- target nucleic acid molecule e.g., one, two, three, four, five, seven, ten, twelve, etc.
- said first and target molecules have one or more homologous sequences (e.g. , one, two, three, four, five, seven, etc.);
- homologous recombination causes transfer of all or a portion of a nucleic acid molecule of the invention containing recombination sites (the first nucleic acid molecule) into one or more positions of the target nucleic acid molecule containing homologous sequences (e.g., one, two, three, four, five, seven, etc.).
- the nucleic acid molecule of the invention comprises at least one selectable marker and at least two sequences which are homologous to the target molecule.
- the first molecule comprises at least two homologous sequences flanking at least one selectable marker.
- the present invention thus facilitates construction of gene targeting nucleic acid molecules or vectors which may be used to knock-out or mutate a sequence or gene of interest (or alter existing sequences, for example to convert a mutant sequence to a wild-type sequence), particularly genes or sequences within a host or host cells such as animals (including animals, such as humans), plants,, insects, bacteria, yeast, and the like or sequences of adventitious agents such as viruses within such host or host cells.
- Such gene targeting may preferably comprise targeting a sequence on the genome of such host cells.
- Such gene targeting may be conducted in vitro (e.g., in a cell-free system) or in vivo (e.g. , in a host cell).
- the invention relates to a method of targeting or mutating a nucleotide sequence or a gene comprising: (a) obtaining at least one nucleic acid molecule of the invention comprising one or more recombination sites (and preferably one or more selectable markers) wherein said molecule comprises one or more nucleotide sequences homologous to the target gene or nucleotide sequence of interest (said one or more homologous sequences preferably flank one or more selectable markers e.g., one, two, three, four, five, seven, ten, etc.) on the molecule of the invention); and
- target genes or nucleotide sequences of interest e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- target genes or nucleotide sequences of interest e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- sites e.g., one, two, three, four, five, seven, ten, etc.
- Such targeting method may cause deletion, activation, inactivation, partial inactivation, or partial activation of the target nucleic acid or gene such that an expression product (typically a protein or peptide) normally expressed by the target nucleic acid or gene is not produced or produced at a higher or lower level or to the extent produced is has an altered protein sequence which may result in more or less activity or in an inactive or partially inactive expression product.
- the selectable marker preferably present on the molecule of the invention facilitates selection of candidates (for example host cells) in which the homologous recombination event was successful.
- the present invention provides a method to produce host cells, tissues, organs, and animals (e.g., transgenic animals) containing the modified nucleic acid or gene produced by the targeting methods of the invention.
- the modified nucleic acid or gene preferably comprises at least one recombination site and/or at least one selectable marker provided by the nucleic acid molecule of the invention.
- the present invention more specifically relates to a method of targeting or mutating a nucleic acid or a gene comprising:
- nucleic acid molecule of the invention comprising one or more recombination sites (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) and at least one selectable marker (e.g., one, two, three, four, five, seven, ten, etc.) flanked by one or more sequences homologous to the target nucleic acid or gene of interest (e.g., one, two, three, four, five, seven, ten, etc.);
- target nucleic acids or genes of interest e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- target nucleic acids or genes of interest e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- selectable markers used in the methods described above are positive selection markers (e.g. , antibiotic resistance markers such
- the invention provides methods for targeting or mutating a target gene or nucleotide sequence comprising, (a) obtaining at least one first nucleic acid molecule comprising one or more recombination sites and one or more selectable markers, wherein the first nucleic acid molecule comprises one or more nucleotide sequences homologous to the target gene or nucleotide sequence; and (b) contacting the first nucleic acid molecule with one or more target genes or nucleotide sequences under conditions sufficient to cause homologous recombination at one or more sites between the target gene or nucleotide sequence and the first nucleic acid molecule, thereby causing insertion of all or a portion of the first nucleic acid molecule within the target gene or nucleotide sequence.
- the first nucleic acid molecule comprises at least one selectable marker flanked by the homologous sequences.
- the selectable marker is flanked by the homologous sequences.
- the target gene or nucleotide sequence is inactivated as a result of the homologous recombination.
- methods of the invention further comprise selecting for a host cell containing the target gene or nucleotide sequence.
- one or more of the one or more nucleotide sequences of the first nucleic acid molecule which are homologous to the target gene or nucleotide sequence will not be 100% identical to the target gene or nucleotide sequence.
- the nucleic acid segments which facilitate homologous recombination need not necessarily share 100% sequence identity.
- these nucleic acid segments will share at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) in their regions of homology.
- nucleic acid segments to facilitate homologous recombination which do not share 100% sequence identity to the nucleic acid with which they are to recombine can be advantageous under a number of instances.
- the homologous nucleic acids correspond to part of a target nucleotide sequence which is a gene and homologous recombination results in the introduction one or more sequence alterations in the target nucleotide sequence.
- the homologous nucleic acids may correspond to a target nucleotide sequence which represents an entire gene. Thus, homologous recombination results in replacement of the target gene.
- Another example of such an instance is where one seeks to perform homologous recombination on an organism which has different nucleotide sequences at the site where homologous recombination is to occur as compared to the one or more homologous nucleotide sequences of the first nucleic acid molecule.
- the differences in these sequences may result, for example, when an organism in which homologous recombination is intended to occur is of a different strain or species than the organism from which the homologous nucleotide sequences of the first nucleic acid molecule are obtained or where the organism has a different genotype at the recombination locus.
- the length of the homologous regions which facilitate recombination can vary in size, but, will generally be at least 15 nucleotides in length (e.g., at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 400 nucleotides, at least 600 nucleotides, at least 800 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3500 nucleotides, at least 4000 nucleotides at least 4500 nucleotides, at least 5000 nucleotides, at least 5500 nucleotides, at least 6000 nucleotides, at least 6000 nucleotides, etc.).
- the invention further provides recombinant host cells produced by the methods described herein, which may be prokaryotic (e.g., bacteria), or eukaryotic (e.g., fungal (e.g., yeasts), plant, or animal (e.g., insect, mammalian including human, etc.) hosts).
- recombination sites introduced into targeted nucleic acids or genes according to the invention may be used to excise, replace, or remove all or a portion of the nucleic acid molecule inserted into the target nucleic acid or gene of interest.
- the invention allows for in vitro or in vivo removal of such nucleic acid molecules and thus may allow for reactivation of the target nucleic acid or gene.
- a selectable marker present on the molecule of the present invention may be removed.
- the present invention also provides methods for cloning the starting or product nucleic acid molecules of the invention into one or more vectors or converting the product molecules of the invention into one or more vectors.
- the starting molecules are recombined to make one or more product molecules and such product molecules are cloned (preferably by recombination) into one or more vectors.
- the starting molecules are cloned directly into one or more vectors such that a number of starting molecules are joined within the vector, thus creating a vector containing the product molecules of the invention.
- the starting molecules are cloned directly into one or more vectors such that the starting molecules are not joined within the vector (i.e., the starting molecules are separated by vector sequences).
- a combination of product molecules and starting molecules may be cloned in any order into one or more vectors, thus creating a vector comprising a new product molecule resulting from a combination of the original starting and product molecules.
- the invention relates to a method of cloning comprising: (a) obtaining at least one nucleic acid molecule of the invention (e.g. , one, two, three, four, five, seven, ten, twelve, etc.) comprising recombination sites; and (b) transferring all or a portion of said molecule into one or more vectors (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, etc.).
- such vectors comprise one or more recombination sites (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) and the transfer of the molecules into such vectors is preferably accomplished by recombination between one or more sites on the vectors (e.g., one, two, three, four, five, seven, ten, etc.) and one or more sites on the molecules of the invention (e.g., one, two, three, four, five, seven, ten, etc.).
- the product molecules of the invention may be converted to molecules which function as vectors by including the necessary vector sequences (e.g. , origins of replication).
- such vectors sequences may be incorporated into the product molecules through the use of starting molecules containing such sequences.
- Such vector sequences may be added at one or a number of desired locations in the product molecules, depending on the location of the sequence within the starting molecule and the order of addition of the starting molecules in the product molecule.
- the invention allows custom construction of a desired vector by combining (preferably through recombination) any number of functional elements that may be desired into the vector.
- the product molecule containing the vector sequences may be in linear form or may be converted to a circular or supercoiled form by causing recombination of recombination sites within the product molecule or by ligation techniques well known in the art.
- circularization of such product molecule is accomplished by recombining recombination sites at or near both termini of the product molecule or by ligating the termini of the product molecule to circularize the molecule.
- linear or circular product molecules can be introduced into one or more hosts or host cells for further manipulation.
- Vector sequences useful in the invention when employed, may comprise one or a number of elements and/or functional sequences and/or sites (or combinations thereof) including one or more sequencing or amplification primers sites (e.g., one, two, three, four, five, seven, ten, etc.), one or more sequences which confer translation termination suppressor activities (e.g., one, two, three, four, five, seven, ten, etc.) such as sequences which encode suppressor tRNA molecules, one or more selectable markers (e.g.
- one, two, three, four, five, seven, or ten toxic genes, antibiotic resistance genes, etc. one or more transcription or translation sites or signals (e.g., one, two, three, four, five, seven, ten, etc.), one or more transcription or translation termination sites (e.g., one, two, three, four, five, seven, ten, twelve, etc.), one or more splice sites (e.g.
- RNA corresponding to recombination sites or protein translated from such sites one or more tag sequences (e.g., HIS6, GST, GUS, GFP, YFP, CFP, epitope tags, etc.), one or more restriction enzyme sites (e.g., multiple cloning sites), one or more origins of replication (e.g., one, two, three, four, five, seven, ten, etc.), one or more recombination sites (or portions thereof) (e.g., one, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), etc.
- tag sequences e.g., HIS6, GST, GUS, GFP, YFP, CFP, epitope tags, etc.
- restriction enzyme sites e.g., multiple cloning sites
- origins of replication e.g., one, two, three, four, five, seven, ten, etc.
- recombination sites or portions thereof
- vector sequences used in the invention may also comprise stop codons which may be suppressed to allow expression of desired fusion proteins as described herein.
- vector sequences may be used to introduce one or more of such elements, functional sequences and/or sites into any of the nucleic acid molecule of the invention, and such sequences may be used to further manipulate or analyze such nucleic acid molecule.
- primer sites provided by a vector preferably located on both sides of the insert cloned in such vector) allow sequencing or amplification of all or a portion of a product molecule cloned into the vector.
- transcriptional or regulatory sequences contained by the vector allows expression of peptides, polypeptides or proteins encoded by all or a portion of the product molecules cloned to the vector.
- genes, portions of genes or sequence tags (such as GUS, GST, GFP, YFP, CFP, His tags, epitope tags and the like) provided by the vectors allow creation of populations of gene fusions with the product molecules cloned in the vector or allows production of a number of peptide, polypeptide or protein fusions encoded by the sequence tags provided by the vector in combination with the product sequences cloned in such vector.
- genes, portions of genes or sequence tags may be used in combination with optionally suppressed stop codons to allow controlled expression of fusion proteins encoded by the sequence of interest being cloned into the vector and the vector supplied gene or tag sequence.
- the vector may comprise one or more recombination sites, one or more stop codons and one or more tag sequences.
- the tag sequences may be adjacent to a recombination site.
- a suppressible stop codon may be incorporated into the sequence of the tag or in the sequence of the recombination site in order to allow controlled addition of the tag sequence to the gene of interest.
- the gene of interest may be inserted into the vector by recombinational cloning such that the tag and the coding sequence of the gene of interest are in the same reading frame.
- the gene of interest may be provided with translation initiation signals (e.g., Shine-Delgarno sequences, Kozak sequences and/or IRES sequences) in order to permit the expression of the gene with a native N-terminal when the stop codon is not suppressed. Further, recombination sites which reside between nucleic acid segments which encode components of fusion proteins may be designed either to not encode stop codons or to not encode stop codons in the fusion protein reading frame.
- the gene of interest may also be provided with a stop codon (e.g., a suppressible stop codon) at the 3'-end of the coding sequence.
- nucleic acid which encodes stop codons can be omitted between each nucleic acid segment and, if desired, nucleic acid which encodes a stop codon can be positioned at the 3' end of the fusion protein coding region.
- a tag sequence may be provided at both the N- and
- the tag sequence at the N-terminal may be provided with a stop codon and the gene of interest may be provided with a stop codon and the tag at the C-terminal may be provided with a stop codon.
- the stop codons may be the same or different.
- the stop codon of the N-terminal tag is different from the stop codon of the gene of interest.
- suppressor tRNAs corresponding to one or both of the stop codons may be provided. When both are provided, each of the suppressor tRNAs may be independently provided on the same vector, on a different vector, or in the host cell genome. The suppressor tRNAs need not both be provided in the same way, for example, one may be provided on the vector contain the gene of interest while the other may be provided in the host cell genome.
- suppression of the stop codon(s) during expression allows production of a fusion peptide having the tag sequence at the N- and/or C-terminus of the expressed protein.
- expression of the sequence of interest without the N- and/or C-terminal tag sequence may be accomplished.
- the invention allows through recombination efficient construction of vectors containing a gene or sequence of interest (e.g. , one, two, three, four, five, six, ten, or more ORF's) for controlled expression of fusion proteins depending on the need.
- the starting nucleic acid molecules or product molecules of the invention which are cloned or constructed according to the invention comprise at least one open reading frame (ORF) (e.g. , one, two, three, four, five, seven, ten, twelve, or fifteen ORFs).
- ORF open reading frame
- Such starting or product molecules may also comprise functional sequences (e.g., primer sites, transcriptional or translation sites or signals, termination sites (e.g. , stop codons which may be optionally suppressed), origins of replication, and the like, and preferably comprises sequences that regulate gene expression including transcriptional regulatory sequences and sequences that function as internal ribosome entry sites (IRES).
- at least one of the starting or product molecules and/or vectors comprise sequences that function as a promoter.
- Such starting or product molecules and/or vectors may also comprise transcription termination sequences, selectable markers, restriction enzyme recognition sites, and the like.
- the starting or product and/or vectors comprise two copies of the same selectable marker, each copy flanked by two recombination sites. In other embodiments, the starting or product and/or vectors comprise two different selectable markers each flanked by two recombination sites. In some embodiments, one or more of the selectable markers may be a negative selectable marker (e.g. , ccdB, kicB, Herpes simplex thymidine kinase, cytosine deaminase, etc.).
- a negative selectable marker e.g. , ccdB, kicB, Herpes simplex thymidine kinase, cytosine deaminase, etc.
- the invention provides methods of cloning nucleic acid molecules comprising (a) providing a first nucleic acid segment flanked by a first and a second recombination site; (b) providing a second nucleic acid segment flanked by a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site; (c) conducting a recombination reaction such that the two nucleic acid segments are recombined into a single nucleic acid molecule; and (d) cloning the single nucleic acid molecule.
- the first recombination site is not capable of recombining with the second and fourth recombination sites and the second recombination site is not capable of recombining with the first and third recombination sites.
- the invention provides a method of cloning comprising providing at least a first nucleic acid molecule comprising at least a first and a second recombination site and at least a second nucleic acid molecule comprising at least a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site and conducting a recombination reaction such that the two nucleic acid molecules are recombined into one or more product nucleic acid molecules and cloning the product nucleic acid molecules into one or more vectors.
- the recombination sites flank the first and/or second nucleic acid molecules.
- the cloning step is preferably accomplished by the recombination reaction of the product molecule into a vector comprising one or more recombination sites, although such cloning steps may be accomplished by standard ligation reactions well known in the art.
- the cloning step comprises conducting a recombination reaction between the sites in the product nucleic acid molecule that did not react in the first recombination reaction with a vector having recombination sites capable of recombining with the unreacted sites.
- the invention provides methods of cloning nucleic acid molecules comprising (a) providing a first nucleic acid segment flanked by at least a first and a second recombination sites and a second nucleic acid segment flanked by at least a third and a fourth recombination sites, wherein none of the recombination sites flanking the first and second nucleic acid segments are capable of recombining with any of the other sites flanking the first and second nucleic acid segments; (b) providing a vector comprising at least a fifth, sixth, seventh and eighth recombination sites, wherein each of the at least fifth, sixth, seventh and eighth recombination sites is capable of recombining with one of the at least first, second, third and/or fourth recombination sites; and (c) conducting a recombination reaction such that the two nucleic acid segments are recombined into the vector thereby cloning the first and the second nucleic acid segments.
- the invention provides a method of cloning comprising providing at least a first nucleic acid molecule comprising at least a first and a second recombination site and at least a second nucleic acid molecule comprising at least a third and a fourth recombination site, wherein none of the first, second, third or fourth recombination sites is capable of recombining with any of the other sites, providing one or more vectors (e.g.
- the method may allow cloning of at least one additional nucleic acid molecule (e.g.
- At least a third nucleic acid molecule wherein said molecule is flanked by a ninth and a tenth recombination site and wherein the vector comprises an eleventh and a twelfth recombination site each of which is capable of recombining with either the ninth or the tenth recombination site.
- the invention also specifically relates to a method of cloning comprising providing a first, a second and a third nucleic acid molecule, wherein the first nucleic acid molecule is flanked by at least a first and a second recombination sites, the second nucleic acid molecule is flanked by at least a third and a fourth recombination sites and the third nucleic acid molecule is flanked by at least a fifth and a sixth recombination sites, wherein the second recombination site is capable of recombining with the third recombination site and the fourth recombination site is capable of recombining with the fifth recombination site, providing a vector having at least a seventh and an eighth recombination sites, wherein the seventh recombination site is capable of reacting with the first recombination site and the eighth recombination site is capable of reacting with the sixth recombination site, and conducting at least one recombination reaction
- the invention provides a method of cloning comprising providing at least a first, a second and a third nucleic acid molecule, wherein the first nucleic acid molecule is flanked by a first and a second recombination site, the second nucleic acid molecule is flanked by a third and a fourth recombination site and the third nucleic acid molecule is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and none of the first, fourth, fifth or sixth recombination sites is capable of recombining with any of the first through sixth recombination sites, providing one or more vectors comprising a seventh and an eighth recombination site flanking at least a first selectable marker and comprising a ninth and a tenth recombination site flanking at least a second selectable marker wherein none of the seventh through tenth recomb
- the selectable markers may be the same or may be different.
- the one or more selectable markers (e.g. , two, three, four, five, seven, etc.) may be negative selectable markers.
- the invention also provides methods of cloning n nucleic acid segments, wherein n is an integer greater than 1, comprising (a) providing n nucleic acid segments, each segment flanked by two recombination sites which do not recombine with each other; (b) providing a vector comprising 2n recombination sites, wherein each of the 2n recombination sites is capable of recombining with one of the recombination sites flanking one of the nucleic acid segments; and (c) conducting a recombination reaction such that the n nucleic acid segments are recombined into the vector thereby cloning the n nucleic acid segments.
- the recombination reaction between the n nucleic acid segments and the vector is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.
- n is 2, 3, 4, or 5.
- the invention generally provides a method of cloning n nucleic acid molecules, wherein n is an integer greater than 1 , comprising the steps of providing n nucleic acid molecules, each molecule comprising at least one and preferably two recombination sites (the two recombination sites preferably flank the n nucleic acid molecule), providing at least one vector comprising one or more recombination sites (and preferably 2n recombination sites) wherein the vector containing recombination sites is capable of recombining with the recombination sites of the n molecules, and conducting a recombination reaction such that the n nucleic acid molecules are inserted into said vectors thereby cloning the n nucleic acid segments.
- the n molecules may be inserted next to or adjoining each other in the vector and/or may be inserted at different positions within the vector.
- the vectors used for cloning according to the invention preferably comprise n copies of the same or different selectable marker, each copy of which is flanked by at least two recombination sites.
- one or more of the selectable markers are negative selectable markers.
- the invention also generally relates to a method of cloning n nucleic acid molecules, wherein n is an integer greater than 1, comprising the steps of providing a 1 st through an n ih nucleic acid molecules, each molecule flanked by at least two recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the i lh segment, n v reacts with one of the recombination sites flanking the r ⁇ j.jth segment and the other recombination site flanking the z th segment, n b reacts with one of the recombination sites flanking the «, + ,th segment, providing a vector comprising at least two recombination sites wherein one of the two recombination sites on the vector react with one of the sites on the 1 st nucleic acid segment and another site on the vector reacts with a recombination site on the « th nucleic acid segment
- nucleic acid molecules/segments cloned by the methods of the invention can be different types and can have different functions depending on the need and depending on the functional elements present.
- at least one of the nucleic acid segments cloned according to the invention is operably linked to a sequence which is capable of regulating transcription (e.g. , a promoter, an enhancer, a repressor, etc.).
- a promoter which is either an inducible promoter or a constitutive promoter.
- RNA produced from the cloned nucleic acid segments results in the production of either a fusion protein or all or part of a single protein.
- at least one of the nucleic acid segments encodes all of part of an open reading frame and at least one of the nucleic acid segments contains a sequence which is capable of regulating transcription (e.g., a promoter, an enhancer, a repressor, etc.).
- at least one of the nucleic acid segments produces a sense RNA strand upon transcription and at least one of the nucleic acid segments produces an antisense
- RNA strand upon transcription the sense RNA and antisense RNA have at least one complementary region and are capable of hybridizing to each other.
- transcription of at least two of the nucleic acid segments results in the production of a single RNA or two separate RNAs.
- these nucleic acid segments may be connected to each other or may be spatially separated within the same nucleic acid molecule.
- the nucleic acid segments comprise nucleic acid molecules of one or more libraries. Further, these libraries may comprise cDNA, synthetic DNA, or genomic DNA. In addition, the nucleic acid molecules of these libraries may encode variable domains of antibody molecules (e.g. , variable domains of antibody light and heavy chains).
- the invention provides screening methods for identifying nucleic acid molecules which encode proteins having binding specificity for one or more antigens and/or proteins having one or more activities (e.g. , secretion from a cell, sub-cellular localization (e.g., localization to the endoplasmic reticulum, the nucleus, mitochondria, chloroplasts, the cell membrane, etc.), ligand binding activity (e.g., small molecules, binding activities for nucleic acids, cell surface receptors, soluble proteins, metal ions, structural elements, protein interaction domains, etc.), enzymatic activity, etc.).
- nucleic acid molecules/segments cloned using methods of the invention may have one or more of the activities referred to above.
- the invention provides methods of cloning at least one nucleic acid molecule comprising (a) providing at least a first, a second and a third nucleic acid segments, wherein the first nucleic acid segment is flanked by at least a first and a second recombination sites, the second nucleic acid segment is flanked by at least a third and a fourth recombination sites and the third nucleic acid segment is flanked by at least a fifth and a sixth recombination sites, wherein the second recombination site is capable of recombining with the third recombination site and none of the first, fourth, fifth or sixth recombination sites is capable of recombining with any of the first through sixth recombination sites; (b) providing a vector comprising at least a seventh and an eighth recombination sites flanking at least a first negative selectable marker and comprising at least a ninth and a tenth recombination sites flanking at least a
- the invention provides methods of cloning at least one nucleic acid molecule comprising (a) providing a first, a second and a third nucleic acid segment, wherein the first nucleic acid segment is flanked by a first and a second recombination site, the second nucleic acid segment is flanked by a third and a fourth recombination site and the third nucleic acid segment is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and the fourth recombination site is capable of recombining with the fifth recombination site; (b) providing a vector comprising a seventh and an eighth recombination site; and (c) conducting at least one recombination reaction such that the second and the third recombination sites recombine and the fourth and the fifth recombination sites recombine and the first and the sixth recombination
- the recombination reaction is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.
- the recombination sites which recombine with each other comprise att sites having identical seven base pair overlap regions.
- the invention provides methods of cloning n nucleic acid fragments, wherein n is an integer greater than 2, comprising (a) providing a 1 st through an n th nucleic acid segment, each segment flanked by two recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the z 'th segment, n i5 reacts with one of the recombination sites flanking the segment and the other recombination site flanking the / th segment reacts with one of the recombination sites flanking the n ⁇ +ith se g men t; ( providing a vector comprising at least two recombination sites, wherein one of the two recombination sites on the vector reacts with one of the sites on the 1 st nucleic acid segment and another site on the vector reacts with a recombination site on the n th nucleic acid segment; and (c) conducting at
- nucleic acid segments are inserted into another nucleic acid molecules. While numerous variations of such methods are possible, in specific embodiments, nucleic acid segments which contain recombination sites having different specificities (e.g. , attLl and attL2) are inserted into a vector which contains more than one set of cognate recombination sites (e.g., ⁇ ttRl and ⁇ ttR2), each set of which flanks negative selection markers. Thus, recombination at cognate sites results can be used to select for nucleic acid molecules which have undergone recombination at one or more of the recombination sites.
- the nucleic acid segments which are inserted into the vector may be the same or different.
- nucleic acid segments may encode expression products or may be transcriptional control sequences.
- vectors of the invention may be used to amplify the copy number or increase expression of encoded products.
- nucleic acid segments when nucleic acid segments are inserted in both direct and inverted orientations, vectors of the invention may be used, for example, to express RNAi, as described elsewhere herein.
- nucleic acid segments encode sequence which regulate transcription (e.g., promoters, enhancers, etc.)
- vectors of the invention may be used to place multiple regulatory elements in operable linkage with nucleic acid that encodes expression products. Vectors of this nature may be used to increased expression of expression products, for example, by providing multiple binding sites for proteins which activate transcription.
- vectors of this nature may be used to decrease expression of expression products, for example, by providing multiple binding sites for proteins which inhibit transcription.
- Vectors of this nature may be used to increased or decrease the expression of expression products, for example, by the expression of multiple copies of nucleic acid molecules which encode factors involved in the regulation of transcription.
- Other embodiments related to the above would be apparent to one skilled in the art.
- the invention provides methods of cloning at least one nucleic acid molecule comprising (a) providing a first population of nucleic acid molecules wherein all or a portion of such molecules are flanked by at least a first and a second recombination sites; (b) providing at least one nucleic acid segment flanked by at least a third and a fourth recombination sites, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site; (c) conducting a recombination reaction such that all or a portion of the nucleic acid molecules in the population are recombined with the segment to form a second population of nucleic acid molecules; and (d) cloning the second population of nucleic acid molecules.
- the recombination reaction is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.
- the second population of nucleic acid molecules encodes a fusion protein.
- the nucleic acid segment encodes a polypeptide which comprises a sequence (preferably an
- N-terminal and/or a C-terminal tag sequence encoding all or a portion of the following: the Fc portion of an immunoglobin, an antibody, a ⁇ -glucuronidase, a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, etc.), a transcription activation domain, a protein or domain involved in translation, protein localization tag, a protein stabilization or destabalization sequence, a protein interaction domains, a binding domain for DNA, a protein substrate, a purification tag (e.g., an epitope tag, maltose binding protein, a six histidine tag, glutathione S-transferase, etc.), and an epitope tag.
- a purification tag e.g., an epitope tag, maltose binding protein, a six histidine tag, glutathione S-transferase, etc.
- the invention provides methods of cloning at least one nucleic acid molecule comprising (a) providing a first population of nucleic acid molecules wherein all or a portion of such molecules are flanked by at least a first and a second recombination site; (b) providing a second population of nucleic acid molecules wherein all or a portion of such molecules are flanked by a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site; (c) conducting a recombination reaction such that all or a portion of the molecules in the first population is recombined with one or more molecules from the second population to form a third population of nucleic acid molecules; and (d) cloning the third population of nucleic acid molecules.
- the recombination reaction is conducted in the presence of one or more recombination proteins under conditions which favor the recombination.
- the invention generally provides methods of joining at least two segments of nucleic acid (including joining populations of nucleic acid molecules), comprising (a) providing at least two segments of nucleic acid (one or both of which may be derived from a population or library of molecules), each segment comprising at least one recombination site capable of recombining with a recombination site present on another (or second) segment; and (b) contacting the segments with one or more recombination proteins under conditions causing recombination between the recombination sites, thereby joining the segments.
- the invention further provides composition comprising the joined nucleic acid segments (or population of segments) prepared by such methods, hosts or host cells comprising such joined nucleic acid segments (which may be populations of host cells or recombinant host cells), and methods of making such hosts or host cells (such as by transforming or transfecting such cells with product molecules of the invention).
- the methods of the invention further comprises inserting the joined nucleic acid segments into one or more vectors.
- the invention also relates to hosts or host cells containing such vectors.
- at least one of the two segments of nucleic acid encodes an expression product (e.g, a selectable marker, an enzyme, a ribozyme, etc.) having one or more identifiable activities.
- at least one of the two segments of nucleic acid contains all or part of an open reading frame (ORF).
- at least one of the two segments of nucleic acid contains a sequence which is capable of regulating transcription (e.g., a promoter, an enhancer, a repressor, etc.).
- one segment encodes an ORF and the other encodes a sequence capable of regulating transcription and/or translation and the recombination reaction allows such sequences to be operably linked.
- one or more of the nucleic acid segments encode a selectable marker or contains an origin of replication.
- some or all of the nucleic acid segments comprise nucleic acid molecules of one or more libraries.
- the one or more libraries comprise polynucleotides which encode variable domains of antibody molecules.
- At least one of the nucleic acid segments encodes a polypeptide linker for connecting variable domains of antibody molecules and/or one or more libraries comprise polynucleotides which encode variable domains of antibody light and heavy chains.
- methods of the invention further comprises at least one screening step to identify nucleic acid molecules which encode proteins having one or more identifiable activities (e.g. , binding specificities for one or more antigens, enzymatic activities, activities associated with selectable markers, etc.).
- the invention can be used to produce modified expression products (by variably linking different segments and/or replacing and/or deleting segments) and analyzing the expression products for desired activities.
- portions of genes and/or a number of genes can be linked to express novel proteins or novel compounds and to select for activities of interest.
- substitution and/or deletions of such linked molecules can also be used to produce altered or modified proteins or compounds for testing.
- biological pathways can be modified by the methods of the invention to, for example, use different enzymes or mutant enzymes in a particular pathway (e.g. , link different enzymes or mutant enzymes which participate in reactions in the same biological pathway).
- modification to biological pathways according to the invention leads to (1) the production of potentially novel compounds such as antibiotics or carbohydrates or (2) unique post-translational modification of proteins (e.g., glycosylation, sialation, etc.).
- the invention also allows for production of novel enzymes by manipulating or changing subunits of multimeric enzyme complexes.
- the invention also provides methods of altering properties of a cell comprising introducing into the cell nucleic acid segments produced by the methods described herein.
- cells altered or produced by methods of the invention are either fungal cells or bacterial cells (e.g., Escherichia coli).
- the invention further provides methods for altering biological pathways and generating new biological pathways.
- genes encoding products involved in the production of a particular pathway e.g., a pathway which leads to the production of an antibiotic
- genes encoding products involved in the production of a particular pathway may be altered using methods of the invention.
- These alterations include the deletion, replacement, and/or mutation of one or more genes which encode products that participate in the pathway.
- regions of genes may be deleted or exchanged following by screening to identify, for example, pathway products having particular features (e.g., a particular methylation pattern).
- genes of different organisms which perform similar but different functions may be combined to produce novel products.
- these products may be identified by screening for specific functional properties (e.g., the ability to inhibit an enzymatic reaction, binding affinity for a particular ligand, antimicrobial activity, antiviral activity, etc.).
- screening methods for identifying compounds which are produced by expression products of nucleic acid molecules of the invention are identified by screening for specific functional properties (e.g., the ability to inhibit an enzymatic reaction, binding affinity for a particular ligand, antimicrobial activity, antiviral activity, etc.).
- nucleic acid segments which encode one or more expression products involved in a particular biological pathway or process have been assembled into one or more nucleic acid molecules, regions of these molecules (e.g., regions which encode expression products) may be deleted or replaced to generate nucleic acid molecules which, for example, express additional expression products, altered expression products, or which do not express one or more expression product involved in the biological pathway or process. Further, nucleic acid segments which encode one or more expression products involved in a particular biological pathway or process may be deleted or inserted as 'a single unit. These methods find application in the production and screening of novel products. In particular, the invention also includes novel products produced by the expression products of nucleic acid molecules described herein.
- the invention provides methods for preparing and identifying nucleic acid molecules containing two or more nucleic acid segments which encode gene products involved in the same biological process or biological pathway, as well as unrelated biological processes or biological pathways, comprising (a) providing a first population of nucleic acid molecules comprising at least one recombination site capable of recombining with other nucleic acid molecules in the first population; (b) contacting the nucleic acid molecules of the first population with one or more recombination proteins under conditions which cause the nucleic acid molecules to recombine and create a second population of nucleic acid molecules; and (c) screening the second population of nucleic acid molecules to identify a nucleic acid molecule which encodes two or more products involved in the same process or pathway.
- the nucleic acid molecules which encodes two or more products involved in the same process or pathway encode two different domains of a protein or protein complex.
- the protein is a single-chain antigen-binding protein.
- the protein complex comprises an antibody molecule or multivalent antigen-binding protein comprising at least two single-chain antigen-binding protein. The invention further provides methods similar to those described above for preparing and identifying nucleic acid molecules containing two or more nucleic acid segments which encode gene products involved in different or unrelated biological processes or biological pathways.
- RNA may be obtained from cells and/or tissues and used to generate cDNA molecules. These cDNA molecules may then be linked to each other and sequenced to identify genes which are expressed in cells and/or tissues, as well as the prevalence of RNA species in these cells and/or tissues.
- the invention provides methods for identifying genes expressed in particular cells and/or tissues and the relative quantity of particular RNA species present in these cells and/or tissues as compared to the quantity of other RNA species.
- Such methods may be used for a variety of applications including diagnostics, gene discovery, the identification of genes expressed in specific cell and/or tissue types, the identification of genes which are over- or under-expressed in particular cells (e.g., cells associated with a pathological condition), the screening of agents to identify agents (e.g., therapeutic agents) which alter gene expression, etc.
- agents e.g., therapeutic agents
- it will often be possible to identify the gene from which a particular RNA species or segment is transcribed by comparison of the sequence data obtained by methods of the invention to nucleic acid sequences cataloged in public databases. Generally, about 10 nucleotides or so of sequence data will be required to identify the gene from which RNA has been transcribed.
- the invention provides methods for determining gene expression profiles in cells or tissues comprising (a) generating at least one population of cDNA molecules from RNA obtained from the cells or tissues, wherein the individual cDNA molecules of the population comprise at least two recombination sites capable of recombining with at least one recombination site present on the individual members of the same or a different population of cDNA molecules; (b) contacting the nucleic acid molecules of (a) with one or more recombination proteins under conditions which cause the nucleic acid molecules to join; and (c) determining the sequence of the joined nucleic acid molecules.
- the joined cDNA molecules are inserted into vectors which contain sequencing primer binding sites flanking the insertion sites.
- the joined cDNA molecules are separated by ⁇ ttB recombination sites.
- the joined cDNA molecules contain between about 10 and about 30 nucleotides which corresponds to the RNA obtained from the cell or tissue.
- the invention provides methods for identifying genes which exhibit alterations in expression as a result of specific stimuli.
- the invention further provides methods for identifying genes involved in cellular metabolism (e.g. , pathological conditions). For example, methods of the invention can be used to determine the expression profile of cells of a particular strain or cells which exhibit an aberrant phenotype. The expression profile of cells of the particular strain or cells which exhibit the aberrant phenotype is compared to the expression profile of cells of another strain or cells which do not exhibit the aberrant phenotype, referred to herein as "reference cells.” By comparison of expression profiles of genes of cells of the particular strain or cells which exhibit the aberrant phenotype to appropriate reference cells, expression characteristics of associated with the strain or aberrant phenotype can be determined.
- reference cells By comparison of expression profiles of genes of cells of the particular strain or cells which exhibit the aberrant phenotype to appropriate reference cells, expression characteristics of associated with the strain or aberrant phenotype can be determined.
- the invention provides diagnostic methods, wherein the gene expression profiles of cells of a patient which exhibit an aberrant phenotype (e.g., cancerous) is compared to the gene expression profiles of cells which do not exhibit the aberrant phenotype (i. e. , reference cells).
- an aberrant phenotype e.g., cancerous
- the invention provides methods for screening therapeutic agents (e.g. , immunostimulatory agent) comprising (a) exposing cells ( ⁇ -g, human cells) to a candidate therapeutic agent, (b) determining the gene expression profile of the exposed cells, (c) comparing the gene expression profile to the gene expression profile of cells which have not been exposed to the candidate therapeutic agent (i. e. , reference cells).
- therapeutic agents e.g. , immunostimulatory agent
- the invention further includes therapeutic agents identified by the methods described above.
- the invention provides a means for attaching or binding through recombination molecules and/or compounds or population of molecules and/or compounds to other molecules, compounds and/or supports (preferably solid or semisolid).
- Suitable molecules and compounds for use in the present invention include, but are not limited to, proteins, polypeptides, or peptides, chemical compounds, drugs, lipids, lipoproteins, carbohydrates, hormones, steroids, antibodies (or portions thereof), antigens, enzymes (e.g., nucleases, polymerases, etc.), polysaccharides, nucleosides and derivatives thereof, nucleotides and derivatives thereof, amino acids and derivatives thereof, fatty acids, receptors, ligands, haptens, small molecules (e.g., activation groups such as -COOH), binding molecules (e.g., biotin, avidin, strepavidin, Protein A, Protein B, etc.), growth factors, metal ions, cytokines, ribozymes, or nucleic acid molecules (e.g., RNA, DNA, DNNRNA hybrids, cDNA or cDNA libraries, double stranded nucleic acids, single stranded nucleic acids, linear
- molecules may be linked to supports either directly or indirectly. Further, molecules may be linked to supports by either covalently or non-covalently.
- molecules may be linked to supports by either covalently or non-covalently.
- one example of the indirect non-covalent linkage of a nucleic acid molecule to a support is where a protein which exhibits high binding affinity for nucleic acid molecules is directly linked to a support. The support containing this protein is then contacted with the nucleic acid molecules under appropriate conditions resulting in the non-covalent attachment of the nucleic acid molecules to the support through the protein.
- This association between nucleic acid molecule/protein interaction can be either sequence specific or non-sequence specific.
- the invention provides supports comprising (either bound or unbound to the support) at least one first nucleic acid molecule, wherein the first nucleic acid molecule comprises one or more recombination sites or portions thereof.
- supports of the invention further comprise at least one second nucleic acid molecule or at least one peptide or protein molecule or other compound bound to the supports through the recombination site on the first nucleic acid molecule.
- the invention also relates to supports of the invention which comprise
- one or more components selected from the group consisting of one or more nucleic acid molecules comprising at least one recombination site, one or more recombination proteins, and one or more peptides or compounds comprising at least one recombination site.
- the invention provides methods for attaching or binding one or more nucleic acid molecules, protein or peptide molecules, or other compounds to supports comprising (a) obtaining at least one nucleic acid molecule, protein or peptide molecule, other compounds, or population of such molecules or compounds comprising at least one recombination site and obtaining supports comprising at least one recombination site; and (b) causing some or all of the recombination sites on the at least one nucleic acid molecule, protein or peptide molecule, other compounds, or population of such molecules or compounds to recombine with all or a portion of the recombination sites comprising the supports.
- the methods further comprise attaching or binding one or more nucleic acid molecules to the supports.
- only one nucleic acid molecule is directly linked to the support.
- the nucleic acid molecules form microarrays.
- the microarrays form a DNA chip.
- the invention further provides supports prepared by the methods described above.
- the support of the invention are either solid or semisolid.
- nucleic acid molecules may be linked to supports either directly or indirectly.
- nucleic acid molecules may be linked to supports either covalently or non-covalently.
- nucleic acid molecules may be linked to supports through linkage to a protein or small molecule (e.g., a molecule having an activation group such as -COOH). Further, nucleic acid molecules may be linked to supports through linkages which are either labile or non-labile.
- the invention provides methods for linking or connecting two or more molecules or compounds of interest, comprising (a) providing at least a first and a second molecule or compound of interest, each of the first and second molecules or compounds of interest comprising at least one recombination site; (b) causing some or all of the recombination sites on the first molecule or compound of interest to recombine with some or all of the recombination sites on the second molecule or compound of interest.
- the methods further comprising attaching nucleic acids comprising recombination sites to the first and the second molecules or compounds of interest.
- at least one of the molecules or compounds of interest comprises a protein or peptide, a nucleic acid, a carbohydrate, a steroid, or a lipid.
- one or more of the compounds and/or molecules of the invention may comprise one or more recombination sites (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or portions thereof.
- recombination sites e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- Such molecules and/or compounds may be unlabeled or detectably labeled by methods well known in the art.
- Detectable labels include, but are not limited to, radioactive labels, mass labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Use of such labels may allow for the detection of the presence or absence of labeled molecules and/or compounds on a support.
- the invention generally relates to attaching to a support any number of molecules and/or compounds or populations of molecules and/or compounds by recombination and the supports made by this method.
- Such compounds and/or molecules can thus be attached to a support or structure via a nucleic acid linker containing a recombination site or portion thereof.
- Such linkers are preferably small (e.g., 5, 20, 30, 50, 100, 200, 300, 400, or 500 base pairs in length).
- the present invention encompasses a support comprising one or a number of recombination sites (or portions thereof) which can be used according to this aspect of the invention.
- nucleic acid molecules, or proteins, peptides and/or other molecules and/or compounds having one or more recombination sites or portions thereof which are to be added or attached or bound to the support are recombined by a recombination reaction with the recombination-site-containing support, thereby creating a support containing one or more nucleic acid molecules, or protein, peptides and/or other molecules and/or compounds of interest.
- the recombination reaction in binding the molecule and/or compound of interest to the support is preferably accomplished in vitro by contacting the support and the molecule and/or compound of interest with at least one recombination protein under conditions sufficient to cause recombination of at least one recombination site on the molecule and/or compound of interest with at least one recombination site present on the support.
- This aspect of the invention is particularly useful in creating arrays of nucleic acids, or proteins and/or other molecules and/or compounds on one or more supports (e.g., two, three, four, five, seven, ten, twelve, etc.) in that it facilitates binding of a number of the same or different nucleic acids, or proteins and/or other molecules and/or compounds of interest through recombination to the support or various parts of the support.
- the invention relates to a method of attaching or binding one or more (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) nucleic acid, or protein molecules and/or other molecules and/or compounds to a support comprising:
- the presence or absence or position of such molecules and/or compounds on the support can be determined (for example by using detectable labels).
- the molecules and/or compounds bound to the support may be further manipulated by well known techniques.
- the invention also allows replacement, insertion, or deletion of one or more molecules and/or compounds contained by the support.
- all or a portion of molecule and/or compound may be removed or replaced with another molecule or compound of interest.
- This process may also be applied to molecules and/or compounds having recombination site which are attached to the support.
- recombination may be used to remove or replace all or a portion of the molecule and/or compound of the interest from the support, in addition to adding all or part of molecules to supports.
- the molecules and/or compounds added to the support or removed from the support may be further manipulated or analyzed in accordance with the invention and as described herein.
- further analysis or manipulation of molecules and/or compounds bound to or removed from the support include sequencing, hybridization (DNA, RNA etc.), amplification, nucleic acid synthesis, protein or peptide expression, protein-DNA interactions (2-hybrid or reverse 2-hybrid analysis), interaction or binding studies with other molecules and/or compounds, homologous recombination or gene targeting, and combinatorial library analysis and manipulation.
- Such manipulation may be accomplished while the molecules and/or compounds are bound to the support or after the molecules and/or compounds are removed from the support.
- any solid or semi-solid supports may be used and sequences containing recombination sites (or portions thereof) may be added by well known techniques for attaching nucleic acids to supports.
- recombination sites may be added to nucleic acid, protein molecules and/or other molecules and/or compounds of interest by techniques well known in the art.
- any wild-type or mutant recombination sites or combinations of the same or different recombination sites may be used for adding and removing molecules and/or compounds of interest to or from a support.
- the invention also relates to any support comprising one or more recombination sites (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or portions thereof and to supports comprising nucleic acid, protein molecules and/or other molecules and/or compounds having one or more recombination sites (or portions thereof) bound to said support.
- recombination sites e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- compositions comprising such supports of the invention.
- Such compositions may further comprise one or more recombination proteins (preferably site specific recombination proteins), suitable buffers (e.g., for causing recombination), nucleic acid, protein molecules and/or other molecules and/or compounds, preferably comprising recombination sites which may be unbound to the support, and any other reagents used for recombining recombination sites according to the invention (and combinations thereof).
- the invention also relates to compositions for use in further manipulating or analyzing the supports of the invention or the nucleic acid or protein molecules or other molecules and/or compounds attached thereto.
- compositions may comprise suitable buffers and enzymes such as restriction enzymes, polymerases, ligases, recombination proteins, and the like.
- the present invention provides a means for attaching or binding one or more (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) molecules and/or compounds or populations of molecules and/or compounds to one or more of the same or different molecules and/or compounds or populations of molecules and/or compounds.
- the invention generally relates to connecting any number of molecules and/or compounds or population of molecules and/or compounds by recombination.
- such linked molecules and/or compounds may be unlabeled or detectably labeled. Further, such linked molecules and/or compounds may be linked to either covalently or non-covalently.
- Suitable molecules and/or compounds include, but are not limited to, those described herein such as nucleic acids, proteins or peptides, chemical compounds, drugs, lipids, lipoproteins, hormones, etc.
- the same molecules and/or compounds, or the same type of molecules and/or compounds e.g., protein-protein, nucleic acid-nucleic acid, etc.
- small molecules and/or proteins may be linked to recombination sites and then linked to each other in various combinations.
- molecules and/or compounds or different types of molecules and/or compounds may be linked through recombination.
- molecules and/or compounds linked through recombination e.g. , protein-protein, protein-ligand, etc.
- the molecules and/or compounds linked through recombination may be attached to a support or structure through recombination as described herein.
- recombination sites (optionally linked to a support) produced are linked by one or more recombination sites (or portions thereof).
- recombination sites may be attached to molecules such as proteins, peptides, carbohydrates, steroids and/or lipids or combinations thereof using conventional technologies and the resulting recombination-site-containing molecules and/or compounds may be linked using the methods of the present invention. Further, the resultant linked molecules and/or compounds may be attached via one or more of the recombination sites to other molecules and/or compounds comprising recombination sites.
- a nucleic acid comprising a recombination site may be attached to a molecule of interest and a second nucleic acid comprising a compatible recombination site may be attached to a second molecule of interest. Recombination between the sites results in the attachment of the two molecules via a small nucleic acid linker.
- the nucleic acid linker may be any length depending on the need but preferably is small (e.g. , from about 5 to about 500 bps in length).
- proteins, peptides, nucleic acids, carbohydrates, steroids and/or lipids or combinations thereof may be attached to proteins, peptides, nucleic acids, carbohydrates, steroids and/or lipids or combinations thereof.
- the present invention provides a method of connecting two or more molecules and/or compounds, comprising the steps of:
- a recombination site may be attached to a molecule of interest using conventional conjugation technology.
- oligonucleotides comprising the recombination site can be synthesized so as to include one or more reactive functional moieties (e.g., two, three, four, five, seven, ten, etc.) which may be the same or different.
- Suitable reactive functional moieties include, but are not limited to, amine groups, epoxy groups, vinyl groups, thiol groups and the like. The synthesis of oligonucleotides comprising one or more reactive functional moieties is routine in the art.
- oligonucleotides comprising one or more reactive functional moieties may be attached to one or more reactive groups (e.g., two, three, four, five, seven, ten, etc.) present on the molecule or compound of interest.
- the oligonucleotides may be attached directly by reacting one or more of the reactive functional moieties with one or more of the reactive functional groups.
- the attachment may be effected using a suitable linking group capable of reacting with one or more of the reactive functional moieties present on the oligonudeotide and with one or more of the reactive groups present on the molecule of interest.
- both direct attachment and attachment through a linking group may be used.
- the reactive functional moieties on the oligonudeotide may be the same or different as the reactive functional moieties on the molecules and/or compounds of interest.
- Suitable reagents and techniques for conjugation of the oligonudeotide to the molecule of interest may be found in Hermanson,
- the present invention also relates to kits for carrying out the methods of the invention, and particularly for use in creating the product nucleic acid molecules of the invention or other linked molecules and/or compounds of the invention (e.g. , protein-protein, nucleic acid-protein, etc.), or supports comprising such product nucleic acid molecules or linked molecules and/or compounds.
- the invention also relates to kits for adding and/or removing and/or replacing nucleic acids, proteins and/or other molecules and/or compounds to or from one or more supports, for creating and using combinatorial libraries of the invention, and for carrying out homologous recombination (particularly gene targeting) according to the methods of the invention.
- kits of the invention may also comprise further components for further manipulating the recombination site-containing molecules and/or compounds produced by the methods of the invention.
- the kits of the invention may comprise one or more nucleic acid molecules of the invention (particularly starting molecules comprising one or more recombination sites and optionally comprising one or more reactive functional moieties), one or more molecules and/or compounds of the invention, one or more supports of the invention and/or one or more vectors of the invention.
- Such kits may optionally comprise one or more additional components selected from the group consisting of one or more host cells (e.g., two, three, four, five etc.), one or more reagents for introducing (e.g.
- molecules or compounds into one or more host cells, one or more nucleotides, one or more polymerases and/or reverse transcriptases (e.g., two, three, four, five, etc.), one or more suitable buffers (e.g. , two, three, four, five, etc.), one or more primers (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), one or more terminating agents (e.g., two, three, four, five, seven, ten, etc.), one or more populations of molecules for creating combinatorial libraries (e.g.
- kits of the invention may also contain directions or protocols for carrying out the methods of the invention.
- kits for joining, deleting, or replacing nucleic acid segments comprising at least one component selected from the group consisting of (1) one or more recombination proteins or compositions comprising one or more recombination proteins, and (2) at least one nucleic acid molecule comprising one or more recombination sites (preferably a vector having at least two different recombination specificities).
- kits of the invention may also comprise one or more components selected from the group consisting of (a) additional nucleic acid molecules comprising additional recombination sites; (b) one or more enzymes having ligase activity; (c) one or more enzymes having polymerase activity; (d) one or more enzymes having reverse transcriptase activity; (e) one or more enzymes having restriction endonuclease activity; (f) one or more primers; (g) one or more nucleic acid libraries; (h) one or more supports; (i) one or more buffers; (j) one or more detergents or solutions containing detergents; (k) one or more nucleotides; (1) one or more terminating agents; (m) one or more transfection reagents; (n) one or more host cells; and (o) instructions for using the kit components.
- kits of the invention may contain one or more recombination proteins selected from the goup consisting of Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ⁇ C31, TndX, XerC, and XerD.
- recombination sites of kits of the invention will generally have different recombination specificities each comprising att sites with different seven base pair overlap regions.
- the first three nucleotides of these seven base pair overlap regions comprise nucleotide sequences selected from the group consisting of ANN, AAC, AAG, AAT, ACA, ACC, ACG, ACT, AGA, AGC, AGG, AGT, ATA, ATC, ATG; ATT, CAN CAC, CAG, CAT, CCA, CCC, CCG, CCT, CGN CGC, CGG, CGT, CTA, CTC, CTG CTT, GAA, GAC, GAG, GAT, GCA, GCC, GCG, GCT, GGA,
- GGC GGG, GGT, GTA, GTC, GTG, GTT, TAA, TAC, TAG, TAT, TCA, TCC, TCG, TCT, TGA, TGC, TGG, TGT, TTA, TTC, TTG, and TTT.
- kits of the invention contain compositions comprising one or more recombination proteins capable of catalyzing recombination between alt sites.
- these compositions comprise one or more recombination proteins capable of catalyzing ⁇ ttB x ⁇ ttP (BP) reactions, ⁇ ttL x ⁇ ttR (LR) reactions, or both BP and LR reactions.
- BP ⁇ ttB x ⁇ ttP
- LR ⁇ ttL x ⁇ ttR
- Nucleic acid libraries supplied with kits of the invention may comprise cDNA or genomic DNA. Further, these libraries may comprise polynucleotides which encode variable domains of antibody light and heavy chains.
- the invention also relates to compositions for carrying out the methods of the invention and to compositions created while carrying out the methods of the invention.
- the invention includes nucleic acid molecules prepared by methods of the invention, methods for preparing host cells which contain these nucleic acid molecules, host cells prepared by these methods, and methods employing these host cells for producing products (e.g., RNA, protein, etc.) encoded by these nucleic acid molecules, products encoded by these nucleic acid molecules (e.g., RNA, protein, etc.).
- compositions, methods and kits of the invention are preferably prepared and carried out using a phage-lambda site-specific recombination system and more preferably with the GATEWAYTM Recombinational Cloning System available from Invitrogen Corp., Life Technologies Division (Rockville, Maryland).
- GATEWAYTM Cloning Technology Instruction Manual (Invitrogen Corp., Life Technologies Division) describes in more detail the systems and is incorporated herein by reference in its entirety.
- Figure 1 is a schematic representation of the basic recombinational cloning reaction.
- Figure 2 is a schematic representation of the use of the present invention to clone two nucleic acid segments by performing an LR recombination reaction.
- Figure 3 is a schematic representation of the use of the present invention to clone two nucleic acid segments by joining the segments using an LR reaction and then inserting the joined fragments into a Destination Vector using a BP recombination reaction.
- Figure 4 is a schematic representation of the use of the present invention to clone two nucleic acid segments by performing a BP reaction followed by an
- FIG. 5 is a schematic representation of two nucleic acid segments having ⁇ ttB sites being cloned by performing a first BP reaction to generate an ⁇ ttL site on one segment and an ⁇ ttR on the other followed by an LR reaction to combine the segments.
- PI, P2, and/or P3 can be oligonucleotides or linear stretches of nucleotides.
- Figure 6 is a schematic representation of the cloning of two nucleic acid segments into two separate sites in a Destination Vector using an LR reaction.
- Figure 7 is a schematic representation of the cloning of two nucleic acid segments into two separate sites in a vector using a BP reaction.
- Figure 8 is a schematic representation of the cloning of three nucleic acid segments into three vectors using BP reactions, cloning the three segments into a single vector using an LR reaction, and generating segments separated by ⁇ ttB sites.
- Figure 9 is a schematic representation of the cloning of three nucleic acid segments into a single vector using a BP reaction and generating segments separated by ⁇ ttR sites.
- Figure 10 is a schematic representation of adding one or more of the same or different molecules (nucleic acid, protein/peptide, carbohydrate, and/or other compounds) to a support (shaded box) by recombination.
- the open boxes represent recombination sites.
- Figure 11 is a schematic representation of joining multiple molecules and/or compounds (A and B). Labels used in this figure correspond to those in Figure 10. The addition of A and B can be simultaneous or sequential.
- Figure 12 is a schematic representation of deleting a portion of a molecule or compound (A) from a support. Labels used in this figure correspond to those in Figure 10.
- Figure 13 is a schematic representation of replacing a portion of a molecule or compound (A) with a second molecule or compound (C). Labels used in this figure correspond to those in Figure 10.
- Figure 14A is a plasmid map showing a construct for providing a C-terminal fusion to a gene of interest. SupF encodes a suppressor function. Thus, when supF is expressed, a GUS-GST fusion protein is produced. In variations of this molecules, GUS can be any gene.
- Figure 14B is a schematic representation of method for controlling both gene suppression and expression.
- the T7 RNA polymerase gene contains one or more (two are shown) amber stop codons (labeled "am”) in place of tyrosine codons.
- Leaky (uninduced) transcription from the inducible promoter makes insufficient supF to result in the production of active T7 RNA polymerase.
- sufficient supF is produced to make active T7 RNA polymerase, which results in increased expression of supF, which results in further increased expression of T7 RNA polymerase.
- the T7 RNA polymerase further induces expression of Gene. Further, expression of supF results in the addition of a C-terminal tag to the Gene expression product by suppression of the intervening amber stop codon.
- Figure 15 is a plasmid map showing a construct for the production of N- and/or C-terminal fusions of a gene of interest. Circled numbers represent amber, ochre, or opal stop codons. Suppression of these stop codons result in expression of fusion tags on the N-terminus, the C-terminus, or both termini. In the absence of suppression, native protein is produced.
- Figure 16 is a schematic representation of the single step insertion of four separate DNA segments into a Destination Vector using LR reactions.
- a first DNA segment having an ⁇ ttL 1 site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a second DNA segment having an ⁇ ttR3 site at the 5' end and an ⁇ ttL4 site at the 3' end.
- the second DNA segment is then linked to a third
- the first, second, third, and fourth DNA segments are inserted into a Destination Vector which contains a ccdB gene flanked by ⁇ ttRl and ⁇ ttR2 sites.
- the inserted DNA segments are separated from each other and vector sequences by ⁇ ttBl, ⁇ ttB3, ⁇ ttB4, ⁇ ttB5, and ⁇ ttB2 sites.
- Figures 17A and 17B show schematic representations of the construction of a lux operon prepared according to the methods set out below in Example 18.
- one or more genes of the operon can be replaced or deleted through recombination to construct one or more modified operons and then tested for activity and/or effect on host cells.
- other genes including variants and mutants
- Figure 18 is a schematic representation of the insertion of six separate DNA segments into a vector using a two step, one vector process.
- a first DNA segment (DNA- A) having an ⁇ ttLl site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a second DNA segment (DNA-B) having an ⁇ ttR3 site at the 5' end and an ⁇ ttL4 site at the 3' end.
- the second DNA segment is then linked to a third DNA segment (DNA-C) having an ⁇ ttR4 site at the 5' end and an ⁇ ttL5 site at the 3' end.
- a fourth DNA segment (DNA-D) having an ⁇ ttRl site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a fifth DNA segment (DNA-E) having an ⁇ ttR3 site at the 5' end and an ⁇ «L4 site at the 3' end.
- the fifth DNA segment is then linked to a sixth DNA segment (DNA-F) having an ⁇ ttR4 site at the 5' end and an ⁇ ttL2 site at the 3' end.
- the two resulting molecules ( . e. , DNA-A-DNA-B-DNA-C and DNA-D-DNA-E-DNA-F) are then inserted into the insertion vector.
- Each of the above reactions is catalyzed by LR CLONASETM.
- An LR reaction is also used to insert the joined DNA segments into a Destination Vector which contains a ccdB gene flanked by ⁇ ttRl and ⁇ ttR2 sites.
- the inserted DNA segments are separated from each other and the vector by ⁇ ttBl, ⁇ ttB3, ⁇ ttB4, ⁇ ttB5, and ⁇ ttB2 sites.
- the assembled segments may be inserted into contiguous or non-contiguous sites.
- Figure 19 is a schematic representation of the insertion of six separate DNA segments into a vector using a two step, two vector process.
- a first DNA segment (DNA- A) having an ⁇ ttB 1 site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a second DNA segment (DNA-B) having an ⁇ ttR3 site at the 5' end and an ⁇ ttL4 site at the 3' end.
- the second DNA segment is then linked to a third DNA segment (DNA-C) having an ⁇ ttR4 site at the 5' end and an ⁇ ttB5 site at the 3' end.
- the linked DNA segments are then inserted into a vector which contains ⁇ ttPl and ⁇ ttP5 sites.
- a fourth DNA segment (DNA- A) having an ⁇ ttB 1 site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a second DNA segment (DNA-B) having an ⁇ ttR3 site
- DNA-D having an ⁇ ttB 5 site at the 5' end and an ⁇ ttL3 site at the 3' end is linked to a fifth DNA segment (DNA-E) having an ⁇ ttR3 site at the 5' end and an ⁇ ttL4 site at the 3' end.
- the fifth DNA segment is then linked to a sixth DNA segment (DNA-F) having an ⁇ ttR4 site at the 5' end and an ⁇ ttB2 site at the 3' end.
- the linked DNA segments are then inserted into a vector which contains ⁇ ttPl and ⁇ ttP2 sites.
- Figure 20A is a schematic representation of an exemplary vector of the invention which contains two different DNA inserts, the transcription of which is driven in different directions by T7 promoters.
- either of DNA- A and/or DNA-B may be in an orientation which results in the production of either sense or anti-sense RNA.
- Figure 20B is a schematic representation of an exemplary vector of the invention which contains one DNA insert, the transcription of which is driven in two different directions by T7 promoters.
- RNA produced by transcription driven by one promoter will be sense RNA and RNA produced by transcription driven by the other promoter will be anti-sense RNA.
- Figure 20C is a schematic representation of an exemplary vector of the invention which contains two different DNA inserts having the same nucleotide sequence (i.e., DNA- A), the transcription of which are driven in different directions by two separate T7 promoters.
- RNA produced by transcription driven by one promoter will be sense RNA and RNA produced by transcription driven by the other promoter will be anti-sense RNA.
- Figure 20D is a schematic representation of an exemplary vector of the invention which contains two DNA inserts having the same nucleotide sequence
- DNA- A in opposite orientations, the transcription of which is driven by one T7 promoter.
- a transcription termination signal is not present between the two copies of DNA-A and the DNA-A inserts.
- Transcription of one segment produces a sense RNA and of the other produces an anti-sense RNA.
- the RNA produced from this vector will undergo intramolecular hybridization and, thus, will form a double-stranded molecule with a hairpin turn.
- Figure 20E is a schematic representation of two exemplary vectors of the invention, each of which contains a DNA insert having the same nucleotide sequence (i.e., DNA-A). Transcription of these inserts results in the production of sense and anti-sense RNA which may then hybridize to form double stranded
- Figure 21 A is a schematic representation of an exemplary vector of the invention which contains three inserts, labeled "promoter,” “coding sequence,” and “Kan r .”
- the inserted promoter drives expression of the coding sequence.
- an inserted DNA segment confers resistance to kanamycin upon host cells which contain the vector.
- vector components e.g. , a selectable marker (for example a kanamycin resistance gene) cassette, an ori cassette, a promoter cassette, a tag sequence cassette, and the like
- a selectable marker for example a kanamycin resistance gene
- FIG 21B is a schematic representation of an exemplary vector of the invention which contains four inserts, labeled “promoter 1 ,” “coding sequence 1 ,” “promoter 2,” and “coding sequence 2.”
- promoter 1 drives expression of coding sequence 1
- promoter 2 drives expression of coding sequence 2.
- Figure 21C is a schematic representation of an exemplary vector of the invention for homologous recombination.
- This vector which contains four inserts, labeled "5' homology,” “NEO,” “DNA-A,” and “3' homology.”
- the 5' and 3' homology regions are homologous to a chromosomal region selected for insertion of a neomycin resistance marker ("NEO") and a DNA segment ("DNA-A").
- Targeting vectors of this type can be designed to insert, delete and/or replace nucleic acid present in targeted nucleic acid molecules.
- Figures 22 A and 22B show a schematic representation of processes for preparing targeting vectors of the invention.
- Figure 23 shows mRNA amplified with random-primed first strand reverse transcription, then random-primed with PCR. These amplification products are split into n pools, and each pool is amplified with random primers with a different pair of ⁇ ttB sites.
- the "R" suffix shows that some of the ⁇ ttB sites can be in inverted orientation.
- ⁇ ttB sites with either the standard or reverse orientations are used in separate pools to generate amplification products where the ⁇ ttB sites are linked in either standard or inverted orientation. When these sites react with inverted ⁇ ttP sites, ⁇ ttR sites are formed in the Entry Clones instead of ⁇ ttL sites.
- Figures 24A-24C show the sequences of a number of att sites (SEQ ID NOs: 1-36) suitable for use in methods and compositions of the invention.
- FIGS 25A-25B show a collection of Entry Clones which contain inserts including, N-terminal tags or sequences (N-tag), open reading frames (ORF), C- terminal tags or sequences (C-tag), selectable markers (amp), origins of plasmid replication (or ⁇ ) and other vector elements (for example a loxP site).
- Each Entry Clone vector element insert is flanked by ⁇ ttL or ⁇ ttR sites such that the vector elements can be linked together and form a new vector construct in an LR Clonase reaction (shown in Figure 25B).
- Figure 26A-26B show a process for constructing ⁇ ttP DONOR plasmids containing ⁇ ttP sites of any orientation and specificity.
- Figure 26A shows four arrangements of ⁇ ttP sites in ⁇ ttP DONOR plasmids consisting of two orientations of direct repeat and two orientations of inverted repeat ⁇ ttP sites.
- the four ⁇ ttP DONOR plasmids shown in Figure 26A can be used as templates for PCR reactions with PCR primers that would anneal specifically to the core of an ⁇ ttP site and thus create an ⁇ ttL or ⁇ ttR site of any desired specificity at the ends of the PCR products.
- PCR primers that would anneal specifically to the core of an ⁇ ttP site and thus create an ⁇ ttL or ⁇ ttR site of any desired specificity at the ends of the PCR products.
- For each new ⁇ ttP DONOR vector to be constructed two such PCR products are generated, one consisting of the plasmid backbone (ori-ka ⁇ ) and a second consisting of the ccdB and cat genes. The PCR products are reacted together in
- Figure 27 A shows a process for linking two nucleic acid segments, A and B.
- the segments are cloned in two similarly configured plasmids.
- Each segment is flanked by two recombination sites.
- One of the recombination sites on each plasmid is capable of reacting with its cognate partner on the other plasmid, whereas the other two recombination sites do not react with any other site present.
- Each plasmid carries a unique origin of replication which may or may not be conditional.
- Each plasmid also carries both positive and negative selectable markers (+smX and smY, respectively) to enable selection against, and for elements linked to a particular marker.
- each plasmid carries a third recombination site (loxP in this example), suitably positioned to enable deletion of undesired elements and retention of desired elements.
- the two plasmids are initially fused at L2 and R2 via a Gateway LxR reaction. This results in the juxtaposition of segments A and B via a B2 recombination site, and the juxtapostion of sml and or ⁇ B via a P2 recombination site.
- the two loxP sites in the backbone that flank a series of plasmid elements are depicted in the second panel. Addition of the Cre protein will resolve the single large plasmid into two smaller ones.
- One of these will be the desired plasmid which carries the linked A and B segments with or/A now linked to sm2 and +sm4.
- the other carries a set of dispensable and/or undesirable elements. Transformation of an appropriate host and subsequent imposition of appropriate genetic selections will result in loss of the undesired plasmid, while the desired plasmid is maintained.
- Figure 27B shows a process for linking two chimeric nucleic acid segments, A-B and C-D, constructed as shown above in Figure 27A.
- the segments are cloned in two similarly configured plasmids. Each segment is flanked by two recombination sites. One of these on each plasmid is capable of reacting with its cognate partner on the other plasmid, whereas the other two recombination sites do not react with any other site present.
- the two plasmids are initially fused at L2 and R2 via a Gateway LxR reaction.
- Gene refers to a nucleic acid which contains information necessary for expression of a polypeptide, protein, or untranslated RNA (e.g. , rRNA, tRNA, anti-sense RNA).
- untranslated RNA e.g. , rRNA, tRNA, anti-sense RNA
- the gene encodes a protein, it includes the promoter and the structural gene open reading frame sequence (ORF), as well as other sequences involved in expression of the protein.
- ORF structural gene open reading frame sequence
- the transcriptional and translational machinery required for production of the gene product is not included within the definition of a gene.
- the gene encodes an untranslated RNA, it includes the promoter and the nucleic acid which encodes the untranslated RNA.
- Structural Gene refers to refers to a nucleic acid which is transcribed into messenger RNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
- Host refers to any prokaryotic or eukaryotic organism that is a recipient of a replicable expression vector, cloning vector or any nucleic acid molecule.
- the nucleic acid molecule may contain, but is not limited to, a structural gene, a transcriptional regulatory sequence (such as a promoter, enhancer, repressor, and the like) and/or an origin of replication.
- host As used herein, the terms "host,” “host cell,” “recombinant host” and “recombinant host cell” may be used interchangeably.
- host cell As used herein, the terms “host,” “host cell,” “recombinant host” and “recombinant host cell” may be used interchangeably.
- host cell See Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).
- transcriptional regulatory sequence refers to a functional stretch of nucleotides contained on a nucleic acid molecule, in any configuration or geometry, that act to regulate the transcription of (1) one or more structural genes (e.g., two, three, four, five, seven, ten, etc.) into messenger RNA or (2) one or more genes into untranslated RNA.
- transcriptional regulatory sequences include, but are not limited to, promoters, enhancers, repressors, and the like.
- a promoter is an example of a transcriptional regulatory sequence, and is specifically a nucleic acid generally described as the 5 '-region of a gene located proximal to the start codon or nucleic acid which encodes untranslated RNA. The transcription of an adjacent nucleic acid segment is initiated at the promoter region.
- a repressible promoter's rate of transcription decreases in response to a repressing agent.
- An inducible promoter's rate of transcription increases in response to an inducing agent.
- a constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.
- insert refers to a desired nucleic acid segment that is a part of a larger nucleic acid molecule.
- the insert will be introduced into the larger nucleic acid molecule.
- the nucleic acid segments labeled ccdB and DNA-A in Figure 2 are nucleic acid inserts with respect to the larger nucleic acid molecule shown therein.
- the insert will be flanked by recombination sites (e.g., at least one recombination site at each end). In certain embodiments, however, the insert will only contain a recombination site on one end.
- target nucleic acid molecule refers to a nucleic acid segment of interest, preferably nucleic acid which is to be acted upon using the compounds and methods of the present invention.
- target nucleic acid molecules preferably contain one or more genes (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or portions of genes.
- Insert Donor refers to one of the two parental nucleic acid molecules (e.g., RNA or DNA) of the present invention which carries the Insert (see Figure 1).
- the Insert Donor molecule comprises the Insert flanked on both sides with recombination sites.
- the Insert Donor can be linear or circular.
- the Insert Donor is a circular nucleic acid molecule, optionally supercoiled, and further comprises a cloning vector sequence outside of the recombination signals.
- a population of Inserts or population of nucleic acid segments are used to make the Insert Donor, a population of Insert Donors result and may be used in accordance with the invention.
- the term "Product” refers to one the desired daughter molecules comprising the A and D sequences which is produced after the second recombination event during the recombinational cloning process (see Figure 1).
- the Product contains the nucleic acid which was to be cloned or subcloned.
- the resulting population of Product molecules will contain all or a portion of the population of Inserts of the Insert Donors and preferably will contain a representative population of the original molecules of the Insert Donors.
- Byproduct refers to a daughter molecule (a new clone produced after the second recombination event during the recombinational cloning process) lacking the segment which is desired to be cloned or subcloned.
- Cointegrate refers to at least one recombination intermediate nucleic acid molecule of the present invention that contains both parental (starting) molecules. Cointegrates may be linear or circular. RNA and polypeptides may be expressed from cointegrates using an appropriate host cell strain, for example E. coli DB3.1 (particularly E. coli LIBRARY EFFICIENCY® DB3.1TM Competent Cells), and selecting for both selection markers found on the cointegrate molecule.
- Recognition Sequence As used herein, the phrase “recognition sequence” refers to a particular sequence to which a protein, chemical compound, DNA, or RNA molecule (e.g.
- a recognition sequence will usually refer to a recombination site.
- the recognition sequence for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence.
- recognition sequences are the ⁇ ttB, ⁇ ttP, ⁇ ttL, and ⁇ ttR sequences which are recognized by the recombinase enzyme ⁇ Integrase.
- ⁇ ttB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region.
- ⁇ ttP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis).
- IHF auxiliary proteins integration host factor
- FIS FIS
- excisionase excisionase
- Such sites may also be engineered according to the present invention to enhance production of products in the methods of the invention.
- engineered sites may lack the PI or HI domains to make the recombination reactions irreversible (e.g., ⁇ ttR or ⁇ ttP)
- such sites may be designated attR or attF to show that the domains of these sites have been modified in some way.
- Recombination proteins includes excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
- recombination proteins include Cre,
- Recombination site refers to a recognition sequence on a nucleic acid molecule which participates in an integration/recombination reaction by recombination proteins. Recombination sites are discrete sections or segments of nucleic acid on the participating nucleic acid molecules that are recognized and bound by a site-specific recombination protein during the initial stages of integration or recombination.
- the recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence. (See Figure 1 of Sauer, B., Curr.
- recognition sequences include the ⁇ ttB, ⁇ ttP, ⁇ ttL, and ⁇ ttR sequences described herein, and mutants, fragments, variants and derivatives thereof, which are recognized by the recombination protein ⁇ Int and by the auxiliary proteins integration host factor
- Recombination sites may be added to molecules by any number of known methods. For example, recombination sites can be added to nucleic acid molecules by blunt end ligation, PCR performed with fully or partially random primers, or inserting the nucleic acid molecules into an vector using a restriction site which flanked by recombination sites.
- Recombinational Cloning refers to a method, such as that described in U.S. Patent Nos. 5,888,732 and 6,143,557 (the contents of which are fully incorporated herein by reference), whereby segments of nucleic acid molecules or populations of such molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo.
- cloning method is an in vitro method.
- Repression Cassette As used herein, the phrase “repression cassette” refers to a nucleic acid segment that contains a repressor or a selectable marker present in the subcloning vector.
- selectable marker refers to a nucleic acid segment that allows one to select for or against a molecule (e.g. , a replicon) or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products which provide resistance against otherwise toxic compounds
- nucleic acid segments that encode products which are otherwise lacking in the recipient cell e.g., tRNA genes, auxotrophic markers
- nucleic acid segments that encode products which suppress the activity of a gene product e.g., phenotypic markers such as ( ⁇ -galactosidase, green fluorescent protein (GFP), yellow flourescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins
- phenotypic markers such as ( ⁇ -galactosidase, green fluorescent protein (GFP), yellow flourescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins
- nucleic acid segments that bind products which are otherwise detrimental to cell survival and/or function (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos.
- nucleic acid segments that bind products that modify a substrate e.g., restriction endonucleases
- nucleic acid segments that can be used to isolate or identify a desired molecule e.g.
- nucleic acid segments that encode a specific nucleotide sequence which can be otherwise non-functional e.g., for PCR amplification of subpopulations of molecules
- nucleic acid segments which when absent, directly or indirectly confer resistance or sensitivity to particular compounds
- nucleic acid segments which when absent, directly or indirectly confer resistance or sensitivity to particular compounds
- nucleic acid segments that encode conditional replication functions e.g. , replication in certain hosts or host cell strains or under certain environmental conditions (e.g., temperature, nutritional conditions, etc.).
- selection scheme refers to any method which allows selection, enrichment, or identification of a desired nucleic acid molecules or host cells contacting them (in particular Product or Product(s) from a mixture containing an Entry Clone or Vector, a Destination
- selection schemes of the invention rely on one or more selectable markers.
- the selection schemes of one embodiment have at least two components that are either linked or unlinked during recombinational cloning.
- One component is a selectable marker.
- the other component controls the expression in vitro or in vivo of the selectable marker, or survival of the cell (or the nucleic acid molecule, e.g., a replicon) harboring the plasmid carrying the selectable marker.
- this controlling element will be a repressor or inducer of the selectable marker, but other means for controlling expression or activity of the selectable marker can be used. Whether a repressor or activator is used will depend on whether the marker is for a positive or negative selection, and the exact arrangement of the various nucleic acid segments, as will be readily apparent to those skilled in the art. In some preferred embodiments, the selection scheme results in selection of or enrichment for only one or more desired nucleic acid molecules (such as Products).
- selecting for a nucleic acid molecule includes (a) selecting or enriching for the presence of the desired nucleic acid molecule (referred to as a "positive selection scheme"), and (b) selecting or enriching against the presence of nucleic acid molecules that are not the desired nucleic acid molecule (referred to as a "negative selection scheme").
- the selection schemes (which can be carried out in reverse) will take one of three forms, which will be discussed in terms of Figure 1.
- the first exemplified herein with a selectable marker and a repressor therefore, selects for molecules having segment D and lacking segment C.
- the second selects against molecules having segment C and for molecules having segment!).
- Possible embodiments of the second form would have a nucleic acid segment carrying a gene toxic to cells into which the in vitro reaction products are to be introduced.
- a toxic gene can be a nucleic acid that is expressed as a toxic gene product (a toxic protein or RNA), or can be toxic in and of itself. (In the latter case, the toxic gene is understood to carry its classical definition of "heritable trait".)
- toxic gene products include, but are not limited to, restriction endonucleases (e.g., Dpn ⁇ , Nla3, etc.); apoptosis-related genes (e.g., ASK1 or members of the bcl-2lced-9 family); retroviral genes; including those of the human immunodeficiency virus (HIV); defensins such as NP-1; inverted repeats or paired palindromic nucleic acid sequences; bacteriophage lytic genes such as those from ⁇ X174 or bacteriophage T4; antibiotic sensitivity genes such as rps ; antimicrobial sensitivity genes such aspheS ; plasmid killer genes' eukaryotic transcriptional vector genes that produce a gene product toxic to bacteria, such as GATA-1; genes that kill hosts in the absence of a suppressing function, e.g., kicB, ccdB, ⁇ X174 E (Liu, Q.
- restriction endonucleases e
- segment/) carries a selectable marker.
- the toxic gene would eliminate transformants harboring the Vector Donor, Cointegrate, and Byproduct molecules, while the selectable marker can be used to select for cells containing the Product and against cells harboring only the Insert Donor.
- the third form selects for cells that have both segments A and D in cis on the same molecule, but not for cells that have both segments in trans on different molecules. This could be embodied by a selectable marker that is split into two inactive fragments, one each on segments A and D.
- the fragments are so arranged relative to the recombination sites that when the segments are brought together by the recombination event, they reconstitute a functional selectable marker.
- the recombinational event can link a promoter with a structural nucleic acid molecule (e.g., a gene), can link two fragments of a structural nucleic acid molecule, or can link nucleic acid molecules that encode a heterodimeric gene product needed for survival, or can link portions of a replicon.
- Site-Specific Recombinase refers to a type of recombinase which typically has at least the following four activities (or combinations thereof): (1) recognition of specific nucleic acid sequences; (2) cleavage of said sequence or sequences; (3) topoisomerase activity involved in strand exchange; and (4) ligase activity to reseal the cleaved strands of nucleic acid.
- Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of sequence specificity for both partners.
- the strand exchange mechanism involves the cleavage and rejoining of specific nucleic acid sequences in the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem. 58:913-949).
- homologous recombination refers to the process in which nucleic acid molecules with similar nucleotide sequences associate and exchange nucleotide strands.
- a nucleotide sequence of a first nucleic acid molecule which is effective for engaging in homologous recombination at a predefined position of a second nucleic acid molecule will therefore have a nucleotide sequence which facilitates the exchange of nucleotide strands between the first nucleic acid molecule and a defined position of the second nucleic acid molecule.
- the first nucleic acid will generally have a nucleotide sequence which is sufficiently complementary to a portion of the second nucleic acid molecule to promote nucleotide base pairing.
- Homologous recombination requires homologous sequences in the two recombining partner nucleic acids but does not require any specific sequences.
- vector refers to a nucleic acid molecule (preferably DNA) that provides a useful biological or biochemical property to an insert. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell.
- a vector can have one or more restriction endonuclease recognition sites (e.g.
- Vectors can further provide primer sites (e.g. , for PCR), transcriptional and/or translational initiation and or regulation sites, recombinational signals, replicons, selectable markers, etc.
- cloning vector can further contain one or more selectable markers (e.g. , two, three, four, five, seven, ten, etc.) suitable for use in the identification of cells transformed with the cloning vector.
- Subcloning vector refers to a cloning vector comprising a circular or linear nucleic acid molecule which includes, preferably, an appropriate replicon.
- the subcloning vector (segment D in Figure 1) can also contain functional and/or regulatory elements that are desired to be incorporated into the final product to act upon or with the cloned nucleic acid insert (segment A in Figure 1).
- the subcloning vector can also contain a selectable marker (preferably DNA).
- Vector Donor refers to one of the two parental nucleic acid molecules (e.g., RNA or DNA) of the present invention which carries the nucleic acid segments comprising the nucleic acid vector which is to become part of the desired Product.
- the Vector Donor comprises a subcloning vector D (or it can be called the cloning vector if the
- Insert Donor does not already contain a cloning vector) and a segment C flanked by recombination sites (see Figure 1 ). Segments C and/or D can contain elements that contribute to selection for the desired Product daughter molecule, as described above for selection schemes.
- the recombination signals can be the same or different, and can be acted upon by the same or different recombinases.
- Vector Donor can be linear or circular.
- primer refers to a single stranded or double stranded oligonudeotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule (e.g., a DNA molecule) .
- the primer may be a sequencing primer (for example, a universal sequencing primer).
- the primer may comprise a recombination site or portion thereof.
- the term "adapter” refers to an oligonudeotide or nucleic acid fragment or segment (preferably DNA) which comprises one or more recombination sites (or portions of such recombination sites) which in accordance with the invention can be added to a circular or linear Insert Donor molecule as well as other nucleic acid molecules described herein. When using portions of recombination sites, the missing portion may be provided by the Insert Donor molecule.
- Such adapters may be added at any location within a circular or linear molecule, although the adapters are preferably added at or near one or both termini of a linear molecule.
- adapters are positioned to be located on both sides (flanking) a particular nucleic acid molecule of interest.
- adapters may be added to nucleic acid molecules of interest by standard recombinant techniques (e.g. , restriction digest and ligation).
- standard recombinant techniques e.g. , restriction digest and ligation
- adapters may be added to a circular molecule by first digesting the molecule with an appropriate restriction enzyme, adding the adapter at the cleavage site and reforming the circular molecule which contains the adapter(s) at the site of cleavage.
- adapters may be added by homologous recombination, by integration of RNA molecules, and the like.
- adapters may be ligated directly to one or more and preferably both termini of a linear molecule thereby resulting in linear molecule(s) having adapters at one or both termini.
- adapters may be added to a population of linear molecules, (e.g. , a cDNA library or genomic DNA which has been cleaved or digested) to form a population of linear molecules containing adapters at one and preferably both termini of all or substantial portion of said population.
- Adapter-primer refers to a primer molecule which comprises one or more recombination sites (or portions of such recombination sites) which in accordance with the invention can be added to a circular or linear nucleic acid molecule described herein. When using portions of recombination sites, the missing portion may be provided by a nucleic acid molecule (e.g., an adapter) of the invention.
- a nucleic acid molecule e.g., an adapter
- Such adapter-primers may be added at any location within a circular or linear molecule, although the adapter-primers are preferably added at or near one or both termini of a linear molecule.
- Such adapter-primers may be used to add one or more recombination sites or portions thereof to circular or linear nucleic acid molecules in a variety of contexts and by a variety of techniques, including but not limited to amplification (e.g., PCR), ligation (e.g., enzymatic or chemical/synthetic ligation), recombination (e.g., homologous or non-homologous (illegitimate) recombination) and the like.
- amplification e.g., PCR
- ligation e.g., enzymatic or chemical/synthetic ligation
- recombination e.g., homologous or non-homologous (illegitimate) recombination
- templates refers to a double stranded or single stranded nucleic acid molecule which is to be amplified, synthesized or sequenced.
- template In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and a second strand is preferably performed before these molecules may be amplified, synthesized or sequenced, or the double stranded molecule may be used directly as a template.
- a primer complementary to at least a portion of the template hybridizes under appropriate conditions and one or more polypeptides having polymerase activity (e.g., two, three, four, five, or seven DNA polymerases and/or reverse transcriptases) may then synthesize a molecule complementary to all or a portion of the template.
- one or more transcriptional regulatory sequences e.g. , two, three, four, five, seven or more promoters
- the newly synthesized molecule may be of equal or shorter length compared to the original template.
- Mismatch incorporation or strand slippage during the synthesis or extension of the newly synthesized molecule may result in one or a number of mismatched base pairs.
- the synthesized molecule need not be exactly complementary to the template.
- a population of nucleic acid templates may be used during synthesis or amplification to produce a population of nucleic acid molecules typically representative of the original template population.
- incorporating means becoming a part of a nucleic acid (e.g., DNA) molecule or primer.
- a library refers to a collection of nucleic acid molecules (circular or linear).
- a library may comprise a plurality of nucleic acid molecules (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, one hundred, two hundred, five hundred one thousand, five thousand, or more), which may or may not be from a common source organism, organ, tissue, or cell.
- a library is representative of all or a portion or a significant portion of the nucleic acid content of an organism (a "genomic” library), or a set of nucleic acid molecules representative of all or a portion or a significant portion of the expressed nucleic acid molecules (a cDNA library or segments derived therefrom) in a cell, tissue, organ or organism.
- a library may also comprise nucleic acid molecules having random sequences made by de novo synthesis, mutagenesis of one or more nucleic acid molecules, and the like.
- Such libraries may or may not be contained in one or more vectors (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.).
- Amplification refers to any in vitro method for increasing the number of copies of a nucleic acid molecule with the use of one or more polypeptides having polymerase activity (e.g., one, two, three, four or more nucleic acid polymerases or reverse transcriptases). Nucleic acid amplification results in the incorporation of nucleotides into a DNA and/or RNA molecule or primer thereby forming a new nucleic acid molecule complementary to a template . The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid replication. DNA amplification reactions include, for example, polymerase chain reaction (PCR) . One PCR reaction may consist of 5 to 100 cycles of denaturation and synthesis of a DNA molecule.
- PCR polymerase chain reaction
- nucleotide refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid molecule (DNA and RNA).
- the term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [ ⁇ SjdATP, 7-deaza-dGTP and 7-deaza-dATP.
- nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP. According to the present invention, a "nucleotide" may be unlabeled or detectably labeled by well known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
- nucleic acid molecule refers to a sequence of contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, or combinations thereof) of any length which may encode a full-length polypeptide or a fragment of any length thereof, or which may be non-coding.
- nucleic acid molecule and “polynucleotide” may be used interchangeably and include both RNA and DNA.
- Oligonudeotide refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides which are joined by a phosphodiester bond between the 3' position of the pentose of one nucleotide and the 5' position of the pentose of the adjacent nucleotide.
- polypeptide refers to a sequence of contiguous amino acids, of any length.
- peptide oligopeptide
- protein may be used interchangeably herein with the term “polypeptide.”
- Hybridization refers to base pairing of two complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double stranded molecule.
- RNA and/or DNA complementary single-stranded nucleic acid molecules
- two nucleic acid molecules may hybridize, although the base pairing is not completely complementary. Accordingly, mismatched bases do not prevent hybridization of two nucleic acid molecules provided that appropriate conditions, well known in the art, are used. In some aspects, hybridization is said to be under
- stringent conditions By “stringent conditions,” as the phrase is used herein, is meant overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
- 5x SSC 750 mM NaCl, 75mM trisodium citrate
- 50 mM sodium phosphate pH 7.6
- 5x Denhardt's solution 10% dextran sulfate
- 20 ⁇ g/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0.1 x SSC at about 65 °C.
- the present invention relates to methods, compositions and kits for the recombinational joining of two or more segments or nucleic acid molecules or other molecules and/or compounds (or combinations thereof).
- the invention also relates to attaching such linked nucleic acid molecules or other molecules and or compounds to one or more supports or structures preferably through recombination sites or portions thereof.
- the invention generally relates to linking any number of nucleic acids or other molecules and/or compounds via nucleic acid linkers comprising one or more recombination sites (e.g. , two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or portions thereof.
- the linked products produced by the invention may comprise any number of the same or different nucleic acids or other molecules and/or compounds, depending on the starting materials.
- Such starting materials include, but are not limited to, any nucleic acids (or derivatives thereof such as peptide nucleic acids (PNAs)), chemical compounds, detectably labeled molecules (such as fluorescent molecules and chemiluminescent molecules), drugs, peptides or proteins, lipids, carbohydrates and other molecules and/or compounds comprising one or more recombination sites or portions thereof.
- PNAs peptide nucleic acids
- detectably labeled molecules such as fluorescent molecules and chemiluminescent molecules
- the joined segments may be inserted into a different nucleic acid molecule such as a vector, preferably by recombinational cloning methods but also by homologous recombination.
- the present invention relates to the construction of nucleic acid molecules (RNA or DNA) by combining two or more segments of nucleic acid (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) by a recombination reaction and inserting the joined two or more segments into a vector by recombinational cloning.
- the timing of the two recombination events i.e., the joining of the segments and the insertion of the segments into a vector
- the timing of the two recombination events is not critical. That is to say, it is not critical to the present invention, for example, whether the two or more nucleic acid segments are joined together before insertion into the vector or whether one recombination site on each segment first reacts with a recombination site on the vector and subsequently the recombination sites on the nucleic acid segments react with each other to join the segments.
- the nucleic acid segments can be cloned in any one or a number of positions within the vector and do not need to be inserted adjacent to each other, although, in some embodiments, joining of two or more of such segments within the vector is preferred.
- recombinational cloning allows efficient selection and identification of molecules (particularly vectors) containing the combined nucleic acid segments.
- two or more nucleic acid segments of interest can be combined and, optionally, inserted into a single vector suitable for further manipulation of the combined nucleic acid molecule.
- At least two nucleic acid segments are contacted with suitable recombination proteins to effect the joining of all or a portion of the two molecules, depending on the position of the recombination sites in the molecules.
- Each individual nucleic acid segment may comprise a variety of sequences including, but not limited to sequences suitable for use as primer sites (e.g., sequences for which a primer such as a sequencing primer or amplification primer may hybridize to initiate nucleic acid synthesis, amplification or sequencing), transcription or translation signals or regulatory sequences such as promoters and or enhancers, ribosomal binding sites, Kozak sequences, start codons, termination signals such as stop codons, origins of replication, recombination sites (or portions thereof), selectable markers, and genes or portions of genes to create protein fusions (e.g., N-terminal or C-terminal) such as GST, GUS, GFP, YFP, CFP, maltose binding protein, 6 histidines (HIS6), epitopes, haptens and the like and combinations thereof.
- sequences suitable for use as primer sites e.g., sequences for which a primer such as a sequencing primer or amplification primer may hybridize to
- the vectors used for cloning such segments may also comprise these functional sequences (e.g., promoters, primer sites, etc.).
- the molecules may be manipulated in a variety of ways, including sequencing or amplification of the target nucleic acid molecule (i.e., by using at least one of the primer sites introduced by the integration sequence), mutation of the target nucleic acid molecule (/. e.
- insertion into another molecule by homologous recombination, transcription of the target nucleic acid molecule, and protein expression from the target nucleic acid molecule or portions thereof (i.e., by expression of translation and/or transcription signals contained by the segments and/or vectors).
- one or more of the nucleic acid segments joined may comprise a nucleic acid library.
- a library may comprise, for example, nucleic acid molecules corresponding to permutations of a sequence coding for a peptide, polypeptide or protein sequence.
- the permutations can be joined to another nucleic acid segment consisting of a single sequence or, alternatively, the second nucleic acid segment may also be a library corresponding to permutation of another peptide, polypeptide or protein sequence such that joining of the two segments may produce a library representing all possible combinations of all the permutations of the two peptide, polypeptide or proteins sequences.
- nucleic acid segments may be contiguous or non-contiguous. Numerous examples of the use of combinatorial libraries are known in the art. (See, e.g., Waterhouse, et al, Nucleic Acids Res., 1993, Vol. 21, No. 9, 2265-2266, Tsurushita, et ⁇ /., Ge «e, 1996, Vol. 172 No. 1,
- nucleic acid segments used in methods and compositions of the invention may contain either
- nucleic acid molecules of a library may be mutated to produce nucleic acid molecules which are, on average, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to corresponding nucleic acid molecules of the original library.
- nucleic acid molecules of a library may be mutated to produce nucleic acid molecules which, encode polypeptides that are, on average, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to polypeptides encoded by corresponding nucleic acid molecules of the original library.
- Recombination sites for use in the invention may be any nucleic acid that can serve as a substrate in a recombination reaction. Such recombination sites may be wild-type or naturally occurring recombination sites, or modified, variant, derivative, or mutant recombination sites. Examples of recombination sites for use in the invention include, but are not limited to, phage-lambda recombination sites (such as ⁇ ttP, ⁇ ttB, ⁇ ttL, and ⁇ ttR and mutants or derivatives thereof) and recombination sites from other bacteriophage such as phi80, P22, P2, 186, P4 and
- PI including lox sites such as loxP and /oxP511).
- Mutated att sites e.g., ⁇ ttB 1-10, ⁇ ttP 1-10, ⁇ ttR 1-10 and ⁇ ttL 1-10) are described in Example 9 below and in previous patent U.S. Appl. No.60/136,744, filed May 28, 1999, and U.S. Appl. No. 09/517,466, filed March 2, 2000, which are specifically incorporated herein by reference.
- Other recombination sites having unique specificity (/. e.
- a first site will recombine with its corresponding site and will not recombine with a second site having a different specificity) are known to those skilled in the art and may be used to practice the present invention.
- Corresponding recombination proteins for these systems may be used in accordance with the invention with the indicated recombination sites.
- FLP/FRT system from Saccharomyces cerevisiae
- the resolvase family e.g., ⁇ , TndX, TnpX, Tn3 resolvase, Hin, Hjc, Gin, SpCCEX , Par A, and Cin
- IS231 other Bacillus thuringiensis transposable elements.
- suitable recombination systems for use in the present invention include the XerC and erD recombinases and the psi, dif ' and cer recombination sites in E. coli.
- Other suitable recombination sites may be found in United States patent no.
- Preferred recombination proteins and mutant, modified, variant, or derivative recombination sites for use in the invention include those described in U.S. Patent Nos. 5,888,732 and 6,143,557, and in U.S. application no. 09/438,358 (filed November 12, 1999), based upon United States provisional application no. 60/108,324 (filed November 13, 1998), and U.S. application no. 09/517,466 (filed March 2, 2000), based upon U.S. provisional application no.
- the inventors have determined that att sites which specifically recombine with other att sites can be constructed by altering nucleotides in and near the 7 base pair overlap region.
- recombination sites suitable for use in the methods, compositions, and vectors of the invention include, but are not limited to, those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region (GCTTTTTTATACTAA (SEQ ID NO:37)), which is identical in all four wild-type lambda att sites, ⁇ ttB, ⁇ ttP, ⁇ ttL and ⁇ ttR (see U.S. Application Nos. 08/663,002, filed June 7, 1996 (now U.S. Patent No.
- Recombination sites suitable for use in the methods, compositions, and vectors of the invention also include those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region (GCTTTTTTATACTAA (SEQ ID NO:37)) which are at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to this 15 base pair core region.
- GCTTTTTTATACTAA SEQ ID NO:37
- nucleic acid molecules suitable for use with the invention also include those which comprising insertions, deletions or substitutions of one, two, three, four, or more nucleotides within the seven base pair overlap region (TTT AT AC, which is defined by the cut sites for the integrase protein and is the region where strand exchange takes place) that occurs within this 15 base pair core region (GCTTTTTTATACTAA (SEQ ID NO:37)).
- TTT AT AC which is defined by the cut sites for the integrase protein and is the region where strand exchange takes place
- mutants, fragments, variants and derivatives include, but are not limited to, nucleic acid molecules in which (1) the thymine at position 1 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (2) the thymine at position 2 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (3) the thymine at position 3 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (4) the adenine at position 4 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; (5) the thymine at position 5 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (6) the adenine at position 6 of the seven
- TTTATAC substitutions made within the first three positions of the seven base pair overlap
- TTTATAC substitutions made in the last four positions
- nucleotide substitutions outside of the seven bp overlap, but elsewhere within the 15 base pair core region do not affect specificity of recombination but do influence the efficiency of recombination.
- nucleic acid molecules and methods of the invention include those which comprising or employ one, two, three, four, five, six, eight, ten, or more recombination sites which affect recombination specificity, particularly one or more (e.g., one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) different recombination sites that may correspond substantially to the seven base pair overlap within the 15 base pair core region, having one or more mutations that affect recombination specificity.
- Particularly preferred such molecules may comprise a consensus sequence such as ⁇ NTAC, wherein " ⁇ " refers to any nucleotide (i.e., may be N, G, T/U or C).
- ⁇ refers to any nucleotide (i.e., may be N, G, T/U or C).
- each att site ( ⁇ ttB, ⁇ ttP, ⁇ ttL and ⁇ ttR) can be divided into functional units consisting of integrase binding sites, integrase cleavage sites and sequences that determine specificity. As discussed below in
- Example 12 specificity determinants are defined by the first three positions following the integrase top strand cleavage site. These three positions are shown with underlining in the following reference sequence: CAACTTTTTTATAC AAAGTTG (SEQ ID ⁇ O:38). Modification of these three positions (64 possible combinations) which can be used to generate att sites which recombine with high specificity with other att sites having the same sequence for the first three nucleotides of the seven base pair overlap region are shown in Table 1.
- the invention further includes nucleic acid molecules comprising one or more (e.g. , one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) nucleotides sequences set out in Table 2.
- the invention provides nucleic acid molecules comprising the nucleotide sequence GAAATAC, GAT AT AC, AC AAT AC, or TGC AT AC.
- the invention will not include nucleic acid molecules which comprise att site core regions set out herein in Figures 24A-24C or in Example 9.
- alterations of nucleotides located 3' to the three base pair region discussed above can also affect recombination specificity.
- alterations within the last four positions of the seven base pair overlap can also affect recombination specificity.
- the invention thus provides recombination sites which recombine with a cognate partner, as well as molecules which contain these recombination sites and methods for generating, identifying, and using these sites. Methods which can be used to identify such sites are set out below in Example 12. Examples of such recombinations sites include att sites which contain 7 base pairs overlap regions which associate and recombine with cognate partners. The nucleotide sequences of specific examples of such 7 base pair overlap regions are set out above in Table 2.
- nucleic acid molecules comprising a nucleotide sequence at least 50% identical, at least 60% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85%o identical, at least 90% identical, or at least 95% identical to the nucleotide sequences of the seven bp overlap regions set out above in Table 2 or the 15 base pair core region shown in SEQ ID NO: 37, as well as a nucleotide sequence complementary to any of these nucleotide sequences or fragments, variants, mutants, and derivatives thereof.
- Additional embodiments of the invention include compositions and vectors which contain these nucleic acid molecules, as well as methods for using these nucleic acid molecules.
- nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations (e.g. , insertions, substitutions, or deletions) per each 100 nucleotides of the reference nucleotide sequence encoding the recombination site.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference ⁇ ttBl nucleotide sequence (SEQ ID NO: 5)
- up to 5% of the nucleotides in the ⁇ ttBl reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the ⁇ ttBl reference sequence may be inserted into the ⁇ ttBl reference sequence.
- These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, a given recombination site nucleotide sequence or portion thereof can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, California) for initial sequence alignment followed by ESEE version 3.0 DNA/protein sequence software (cabot@trog.mbb.sfu.ca) for multiple sequence alignments.
- DNAsis software Haitachi Software, San Bruno, California
- ESEE version 3.0 DNA/protein sequence software cabot@trog.mbb.sfu.ca
- such determinations may be accomplished using the BESTFIT program (Wisconsin Sequence Analysis Package, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711), which employs a local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of homology between two sequences.
- the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
- the invention further provides, in one aspect, methods for constructing and/or identifying recombination sites suitable for use with nucleic acid molecules of the invention, as well as recombination sites constructed and/or identified by these methods.
- the invention provides methods for constructing and/or identifying recombination sites which are capable of recombining with other recombination sites.
- the invention provides methods for constructing recombination sites and identifying whether these recombination sites recombine with other recombination sites.
- Recombination sites which are screened for recombination activity and specificity can be constructed by any number of means, including site-directed mutagenesis and random nucleic acid synthesis.
- the invention further provides "single use" recombination sites which undergo recombination one time and then either undergo recombination with low frequency (e.g. , have at least five fold, at least ten fold, at least fifty fold, at least one hundred fold, or at least one thousand fold lower recombination activity in subsequent recombination reactions) or are essentially incapable of undergo recombination.
- the invention also provides methods for making and using nucleic acid molecules which contain such single use recombination sites and molecules which contain these sites. Examples of methods which can be used to generate and identify such single use recombination sites are set out below.
- the ⁇ tt system core integrase binding site comprises an interrupted seven base pair inverted repeat having the following nucleotide sequence: > ⁇ caactttnnnnnnnaaa ⁇ tt ⁇ (SEQ ID NO:39), as well as variations thereof which can comprise either perfect or imperfect repeats.
- the repeat elements can be subdivided into two distal and/or proximal
- domains composed of caac/gttg segments (underlined), which are distal to the central undefined sequence (the nucleotides of which are represented by the letter “n"), and ttt/aaa segments, which are proximal to the central undefined sequence.
- Alterations in the sequence composition of the distal and/or proximal domains on one or both sides of the central undefined region can affect the outcome of a recombination reaction.
- the scope and scale of the effect is a function of the specific alterations made, as well as the particular recombinational event (e.g. , LR vs. BP reactions).
- single use recombination sites may be prepared by altering nucleotides in the seven base pair inverted repeat regions which abut seven base pair overlap regions of ⁇ tt sites.
- This region is represented schematically as: CAAC TTT [Seven Base Pair Overlap Region] AAA GTTG.
- nucleotides of the sequences CAACTTT or AAAGTTG may be substituted with other nucleotides or deleted altogether.
- These seven base pair inverted repeat regions represent complementary sequences with respect to each other.
- alterations may be made in either seven base pair inverted repeat region in order to generate single use recombination sites.
- DNA is double stranded and one seven base pair inverted repeat region is present, the other seven base pair inverted repeat region will also be present on the other strand.
- examples of seven base pair inverted repeat regions which can form single use recombination sites include, but are not limited to, nucleic acid molecules in which (1) the cytosine at position 1 of the seven base pair inverted repeat region has been deleted or substituted with a guanine, adenine, or thymine; (2) the adenine at position 2 of the seven base pair inverted repeat region has been deleted or substituted with a guanine, cytosine, or thymine; (3) the adenine at position 3 of the seven base pair inverted repeat region has been deleted or substituted with a guanine, cytosine, or thymine; (4) the cytosine at position 4 of the seven base pair inverted repeat region has been deleted or substituted with a guanine, adenine, or thymine; (5) the thymine at position 5 of the seven base pair inverted repeat region has been deleted or substituted with a guanine, cytosine
- nucleotide sequences which form single use recombination sites may also be prepared by combining a nucleotide sequence set out in Table 4, Section 1 , with a nucleotide sequence set out in Table 4, Section 2.
- Single use recombination sites may also be prepared by the insertion of one or more (e.g., one, two, three, four, five six, seven, etc.) nucleotides internally within these regions.
- the altered sequence will be located proximally to the nucleic acid segment.
- nucleic Acid Segment 3' caac ttt [Seven Base Pair Overlap Region] AAA GTTG, the lower case nucleotide sequence which represent a seven base pair inverted repeat region (i.e., caac ttt) will generally have a sequence altered by insertion, deletion, and/or substitution to form a single use recombination site when one seeks to prevent recombination at the 3 ' end (/. e. , proximal end with respect to the nucleic acid segment) of the nucleic acid segment shown.
- a single recombination reaction can be used, for example, to integrate the nucleic acid segments into another nucleic acid molecule, then the recombination site becomes effectively non-functional, preventing the site from engaging in further recombination reactions.
- single use recombination sites can be position at both ends of a nucleic acid segment so that the nucleic acid segment can be integrated into another nucleic acid molecule, or circularized, and will remain integrated, or circularized even in the presence of recombinases.
- a number of methods may be used to screen potential single use recombination sites for functional activity (e.g. , undergo one recombination event followed by the failure to undergo subsequent recombination events).
- a first recombination reaction may be performed to generate a plasmid in which a negative selection marker is linked to one or more potentially defective recombination sites.
- the plasmid may then be reacted with another nucleic acid molecule which comprises a positive selection marker similarly linked to recombination sites.
- this selection system is designed such that molecules which recombine are susceptible to negative selection and molecules which do not recombine may be selected fro by positive selection. Using such a system, one may then directly select for desired single use core site mutants.
- screening assays may be designed which achieve the same results as those described above. In many instances, these assays will be designed so that an initial recombination event takes place and then recombination sites which are unable to engage in subsequent recombination events are identified or molecules which contain such recombination sites are selected for. A related screening assay would result in selection against nucleic acid molecule which have undergone a second recombination event. Further, as noted above, screening assays can be designed where there is selection against molecules which have engaged in subsequent recombination events and selection for those which have not engaged in subsequent recombination events.
- Single use recombination sites are especially useful for either decreasing the frequency of or preventing recombination when either large number of nucleic acid segments are attached to each other or multiple recombination reactions are performed.
- the invention further includes nucleic acid molecules which contain single use recombination sites, as well as methods for performing recombination using these sites.
- the invention provides a modular system for constructing nucleic acid molecules having particular functions or activities.
- the invention further provides methods for combining populations of nucleic acid molecules with one or more known or unknown target sequences of interest (e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) or with other populations of nucleic acid molecules (known or unknown), thereby creating populations of combinatorial molecules (e.g., combinatorial libraries) from which unique and/or novel molecules (e.g., hybrid molecules) and proteins or peptides encoded by these molecules may be obtained and further analyzed.
- target sequences of interest e.g., two, three, four, five seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.
- combinatorial molecules e.g., combinatorial libraries
- unique and/or novel molecules e.g., hybrid molecules
- proteins or peptides encoded by these molecules may be obtained and further analyzed.
- the present invention also includes methods for preparing vectors containing more than one nucleic acid insert (e.g. , two, three, four, five, six, eight, ten, twelve, fifteen, twenty, thirty, forty, fifty, etc. inserts).
- vectors of the invention are prepared as follows. Nucleic acid molecules which are to ultimately be inserted into the Destination Vector are obtained (e.g., purchased, prepared by PCR or by the preparation of cDNA using reverse transcriptase). Suitable recombination sites are either incorporated into the 5' and 3' ends of the nucleic acid molecules during synthesis or added later.
- these inserts can be inserted into a vector in either one reaction mixture or a series of reaction mixtures.
- multiple nucleic acid segments can be linked end to end and inserted into a vector using reactions performed, for example, in a single reaction mixture.
- the nucleic acid segments in this reaction mixture can be designed so that recombination sites on their 5' and 3' ends result in their insertion into a Destination Vector in a specific order and a specific 5' to 3' orientation.
- nucleic acid segments can be designed so that they are inserted into a Destination Vector without regard to order, orientation (i.e., 5' to 3' orientation), the number of inserts, and/or the number of duplicate inserts. Further, in some instances, one or more of the nucleic acid segments will have a recombination site on only one end. Also, if desired, this end, or these ends, may be linked to other nucleic acid segments by the use of, for example, ligases or topoisomerases.
- a linear nucleic acid molecule with an ⁇ ttRl site on its 5' terminus can be recombined with a Destination Vector containing a ccdB gene flanked by an ⁇ ttLl site and an ⁇ ttL2 site.
- the Destination Vector Before, during, or after an LR reaction, the Destination Vector can be cut, for example, by a restriction enzyme on the side of the ⁇ ttR2 site which is opposite to the ccdB gene.
- the Destination Vector will be linear after being cut and undergoing recombination.
- the ⁇ ttRl site of the nucleic acid molecule will undergo recombination with the ⁇ ttLl site of the Destination Vector to produce a linear vector which contains the nucleic acid molecule.
- the resulting linear product can then be circularized using an enzyme such as a ligase or topoisomerases.
- a first DNA segment having an ⁇ ttLl site at the 5' end and an ⁇ ttL3 site at the 3' end is attached by recombination to a second DNA segment having an ⁇ ttR3 site at the 5' end and an ⁇ ttL4 site at the 3' end.
- a third DNA segment having an ⁇ ttR4 site at the 5' end and an ⁇ ttL5 site at the 3' end is attached by recombination with the ⁇ ttL4 site on the 3' end of the second DNA segment.
- a fourth DNA segment having an ⁇ ttR5 site at the 5' end and an ⁇ ttL2 site at the 3' end is attached by recombination with the ⁇ ttL5 site on the 3' end of the third DNA segment.
- the Destination Vector contains an ⁇ ttRl site and an ⁇ ttR2 site which flanks a ccdB gene.
- the first, second, third, and fourth DNA segments are inserted into the insertion vector but are flanked or separated by ⁇ ttB 1 , ⁇ ttB3, ⁇ ttB4, ⁇ ttB5, and ⁇ ttB2 sites.
- a similar process involving assembly of the lux operon is shown in Figures 17A-17B and described below in Example 18.
- FIG. 18 One example of a multi-reaction process for inserting multiple DNA segments into a vector is shown in Figure 18. In this exemplary embodiment, three DNA segments recombine with each other in two separate reaction mixtures. The products generated in these mixtures are then mixed together under conditions which facilitate both recombination between the products of the two reaction mixtures and insertion of the linked product into a vector (e.g., a
- This embodiment has the advantages that the (1) DNA segments can be inserted directly into a Destination Vector without prior insertion into another vector, and (2) the same att sites, as well as other recombination sites, can be used to prepare each of the linked DNA segments for insertion into the vector.
- nucleic acid segments can be connected randomly, or in a specified order, both with or without regard to 5' to 3' orientation of the segments.
- identical copies of one or more nucleic acid segments can be incorporated into another nucleic acid molecule.
- the invention also provides nucleic acid molecules which contain multiple copies of a single nucleic acid segment.
- recombination sites positioned at the 5' and 3' ends of these segments can be used to determine the exact number of identical nucleic acid segments which are connected and then inserted, for example, into a vector.
- Such vectors may then be inserted into a host cell where they can, for example, replicate autonomously or integrate into one or more nucleic acid molecules which normally reside in the host cell (e.g., integrate by site-specific recombination or homologous recombination).
- Nucleic acid molecules which contain multiple copies of a nucleic acid segment may be used, for example, to amplify the copy number of a particular gene.
- the invention also provides methods for gene amplification, nucleic acid molecules which contain multiple copies of a nucleic acid segment, and host cells which contain nucleic acid molecules of the invention.
- nucleic acid segments can be connected using processes of the invention. Recombination sites can be positioned on these segments, for example, such that the segments alternate upon attachment (e.g., Segment A+Segment B+Segment A+Segment B, etc.).
- a nucleic acid molecule having such a structure will be especially useful for when one seeks to use increased copy number of a nucleic acid to increase the amount of expression product produced.
- “Segment A” can be, for example, a nucleic acid molecule comprising an inducible promoter
- Segment B can be, for example, a nucleic acid molecule comprising an ORF.
- cells can be prepared which contain the above construct and do not express substantial quantities of the product of Segment B in the absence of the inducing signal but produce high levels of this product upon induction.
- Such a system will be especially useful when the Segment B expression product is toxic to cells.
- the methods set out above can be used for the construction and maintenance of cells which contain Segment B in the absence of deleterious effects resulting from the Segment B expression product.
- induction of expression of the ORF residing in Segment B can then be used, for example, to transiently produce high levels of the Segment B expression product.
- recombination sites can be chosen so as to link nucleic acid segments in one reaction and not engage recombination in later reactions.
- a series of concatamers of ordered nucleic acid segments can be prepared using ⁇ ttL and ⁇ ttR sites and LR ClonaseTM. These concatamers can then be connected to each other and, optionally, other nucleic acid molecules using another LR reaction. Numerous variations of this process are possible.
- single use recombination sites may be used to prevent nucleic acid segments, once incorporated into another nucleic acid molecule, from engaging in subsequent recombination reactions.
- the use of single use recombination sites allows for the production of nucleic acid molecules prepared from an essentially limitless number of individual nucleic acid segments.
- the invention further provides method for combining nucleic acid molecules in a single population with each other or with other molecules or populations of molecules, thereby creating populations of combinatorial molecules from which unique and/or novel molecules (e.g. , hybrid molecules) and proteins or peptides encoded by these molecules may also be obtained and further analyzed.
- the invention further provides methods for screening populations of nucleic acid molecules to identify those which have particular activities or which encode expression products (e.g., RNAs or polypeptides) which have particular activities.
- methods of the invention can be used to combine nucleic acid segments which encode functional domains
- nucleic acid segments which contain transcriptional regulatory sequences can be identified by the following methods.
- the nucleic acid molecules of a genomic DNA library are modified to contain recombination sites on their 5' and 3' termini. These nucleic acid molecules are then inserted into a Destination Vector such that they are located 5' to a selectable marker.
- expression of the selectable marker will occur in vectors where the marker is in operable linkage with a nucleic acid molecule which activates its transcription.
- the invention thus further provides isolated nucleic acid molecules which are capable of activating transcription. In many instances, these nucleic acid molecules which activate transcription will be identified using methods and/or compositions of the invention.
- tissue-specific transcriptional regulatory sequences activate gene expression in a tissue-specific manner
- methods of the invention can be used to identify tissue-specific transcriptional regulatory sequences. For example, when one seeks to identify transcriptional regulatory sequences which activate transcription in a specific cell or tissue type, the above screening process can be performed in cells of that cell or tissue type. Similarly, when one seeks to identify regulatory sequences which activate transcription in cells at a particular time, at a particular stage of development, or incubated under particular conditions (e.g. , at a particular temperature), the above screening process can be performed in cells at an appropriate time, at the particular stage of development or incubated under the particular conditions.
- the transcriptional regulatory sequences can then be tested to determine if it is capable of activating transcription in other cells types or under conditions other than those which resulted in its identification and/or selection.
- the invention provides methods for constructing and/or identifying transcriptional regulatory sequences, as well as nucleic acid molecules which contain transcriptional regulatory sequences identified by methods of the invention in operable linkage with nucleic acid segments which encode expression products and methods for preparing such molecules.
- the invention further includes methods for identifying nucleic acid molecules which contain origins of replication, as well as nucleic acid molecules which contain origins of replication identified by methods of the invention and methods for preparing such molecules.
- the invention is thus particularly suited for the construction of combinatorial libraries.
- methods of the invention can also be used to "shuffle" nucleic acid molecules which encode domains and regions of proteins to generate new nucleic acid molecules which can be used to express proteins having specific properties or activities.
- nucleic acid segments which encode portions of proteins are joined and then screened for one or more properties or activities.
- the nucleic acid segments in these combinatorial libraries may be prepared by any number of methods, including reverse transcription of mRNA. Altered forms of the nucleic acid segments in these libraries may be generated using methods such as error prone PCR. In many applications, it will be desirable for the nucleic acid segments in these libraries to encode subportions of protein. When this is the case, the methods can be adjusted to generate populations of nucleic acid segments the majority of which do not contain full length ORFs. This can be done, for example, by shearing the cDNA library and then separating the sheared molecules (e.g., using polyacrylamide or agarose gel electrophoresis).
- Fragments between, for example, 300 and 600 nucleotides in length may then be recombined and inserted into a vector in operable linkage with a transcriptional regulatory sequence.
- Polypeptide expression products of the individual members of such a combinatorial library may then be screened to identify those with particular properties or activities.
- the invention further provides methods for producing combinatorial libraries generated using exon nucleic acid derived from genomic DNA.
- Intron/exon splice boundaries are known in the art; thus the locations of exons in genomic DNA can be identified using routine, art-known methods without undue experimentation. Further, primers corresponding to intron/exon splice boundaries can be used to generate nucleic acid molecules which correspond to exon sequences. Further, these nucleic acid molecules may then be connected to each other to generate combinatorial libraries comprising nucleic acid molecules which correspond to exon sequences. For example, primers corresponding to intron/exon splice boundaries can be used to generate nucleic acid molecules which correspond to exon sequences using PCR.
- Recombination sites may then be added to the termini of the resulting PCR products using ligases or amplifying the sequences using primers containing recombination sites.
- the PCR products may then be connected to each other using recombination reactions and inserted into an expression vector.
- the resulting combinatorial library may then be screened to identify nucleic acid molecules which, for example, encode polypeptides having particular functions or activities. Further, recombination sites in expression products (e.g., RNA or protein) of nucleic acid molecules of the combinatorial library can be removed by splicing as described elsewhere herein.
- nucleic acid molecules used to produce combinatorial libraries may be mutated to produce nucleic acid molecules which are, on average, at least 50%, at least 55%, at least 60%), at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90%), at least 95%, at least 96%, at least 91%, at least 98%, or at least 99% identical to the corresponding original nucleic acid molecules.
- nucleic acid molecules used to produce combinatorial libraries may be mutated to produce nucleic acid molecules which, encode polypeptides that are, on average, are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%), at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to polypeptides encoded by the corresponding original nucleic acid molecules.
- the invention provides methods for generating and identifying dominant/negative suppressors of biological processes or biological pathways.
- combinatorial libraries described above can be screened for dominant/negative activity.
- dominant/negative activity results in the suppression of a biological process or biological pathway.
- dominant/negative suppressors exhibit their affects through interaction with cellular components.
- many dominant/negative suppressors contain domains having binding activities associated with one or more cellular proteins but do not have other activities associated with the cellular proteins.
- dominant/negative suppressors upon expression in a cell, dominant/negative suppressors generally interact with one or more cellular ligands and block activation by cellular proteins.
- ligand sequestration one mechanism by which dominant/negative suppressors are believed to interfere with normal cellular processes.
- Dominant/negative activity can be conferred by mutations in a wild-type protein such as an alteration of a single amino acid residue or a deletion of an entire region of the protein.
- a wild-type protein such as an alteration of a single amino acid residue or a deletion of an entire region of the protein.
- Oury eta J. Biol. Chem. 275:22611-22614 (2000), for example, describe a dominant/negative receptor where dominant/negative activity results from the deletion of a single amino acid residue.
- Protein fragments can also have dominant/negative activity.
- McNellis et al, Plant Cell 8: 1491 - 1503 (1996) describe an N-terminal fragment of constitutive photomorphogenic 1 protein (COP 1 ) which has dominant/negative activity when expressed in Arabidopsis seedlings.
- COP 1 constitutive photomorphogenic 1 protein
- EPAS1 endothelial PAS domain protein 1
- Maemura et al demonstrated that expression of the EPAS1 mutant in cells inhibits induction of VEGF mRNA production, an activity associated with wild-type EP AS 1.
- the invention also provides methods for identifying nucleic acid molecules which encode polypeptides having particular functions or activities, as well as nucleic acid molecules produced by these methods, expression products of these nucleic acid molecules, and host cells which contain these nucleic acid molecules.
- functions or activities include secretion from cells, enzymatic activities, ligand binding activities (e.g., binding affinity for metal ions, cell surface receptors, nucleic acids, soluble proteins), and the ability to target the expression product to a sub-cellular localization (e.g., localization to mitochondria, chloroplasts, endoplasmic reticulum, etc.).
- Assays for identifying these nucleic acid molecules will generally be designed to identify the function of activity associated with the polypeptide.
- nucleic acid molecules which encode polypeptides having regions which interact with other polypeptides.
- One example of such a method involves the use of two hybrid assays. (See, e.g., Fields et al, U.S. Patent No. 5,667,973, the entire disclosure of which is incorporated herein by reference.) More specifically, nucleic acid molecules can be prepared using methods of the invention which encode a fusion protein between a polypeptide (e.g., a Gal4N-terminal domain) that exhibits a particular function when in close proximity with another polypeptide (e.g., a Gal4C-terminal domain) and protein or region of a protein for which a ligand is sought.
- a polypeptide e.g., a Gal4N-terminal domain
- another polypeptide e.g., a Gal4C-terminal domain
- nucleic acid molecules are then prepared which encode fusions between the other polypeptide referred to in the previous sentence and protein segments encoded by a combinatorial library.
- nucleic acid segments in the combinatorial library which encode desired ligands can be identified by screening for activities associated conferred by bringing the two polypeptides into close proximity with each other.
- Phage and bacterial surface display libraries may also be generated by methods of the invention to identify domains which have particular functional activities (e.g., binding activity for a particular ligand).
- Kim et al Appl. Environ. Microbiol. 55:788-793 (2000), describe a bacterial surface display method for the selectively screening for improved variants of carboxymethyl cellulase (CMCase).
- CMCase carboxymethyl cellulase
- a library of mutated CMCase genes is generated by DNA shuffling and fused to the ice nucleation protein (Inp) gene, which results in the fusion proteins being displayed on the
- the invention thus provide methods for identifying nucleic acid segments which encode proteins or protein regions that interact with other proteins or have particular functional activities, as well as nucleic acid segments identified by such methods and polypeptide expression products of these nucleic acid segments.
- methods of the invention involve generating combinatorial libraries and screening these libraries to identify individual nucleic acid molecules which encode expression products that interact with a particular protein or have a particular activity.
- the combinatorial libraries described above will encode fusion proteins.
- methods of the invention can be used to prepare and identify nucleic acid molecules which encode proteins and protein variants having particular properties, functions or activities.
- a protein property which is readily assayable is solubility. For example, fluorescence generated by GFP is quenched when an insoluble GFP fusion protein is produced. Further, alterations in a relatively small number of amino acid residues of a protein (e.g., one, two, three, four, etc.), when appropriately positioned, can alter the solubility of that protein.
- combinatorial libraries which express GFP fusion proteins can be used to isolate proteins and protein variants which have altered solubility.
- a combinatorial library designed to express GFP fused with variants of a single, insoluble polypeptide can be used to isolate nucleic acid molecules which encode soluble variants of the polypeptide.
- nucleic acid molecules which contain two or more nucleic acid segments, wherein expression one nucleic acid segment is facilitated by the expression product of one of the other nucleic acid segments.
- one nucleic acid segment may be operably linked to a T7 polymerase promoter and another nucleic acid segments encodes a T7 polymerase.
- the nucleic acid segment operably linked to the T7 polymerase promoter will be expressed upon expression of the T7 polymerase.
- nucleic acid encoding components or having particular activities referred to above can reside in a vector into which one or more the nucleic acid segments are inserted.
- Methods of the invention can also be used to construct nucleic acid molecules which encode more than one subunit of a multi-subunit enzyme. Further, expression of each of the subunits of this enzyme may be regulated by the same promoter or different promoter. When the same promoter is used to drive expression of nucleic acid which encode two or more proteins, the mRNA may contain, for example one or more internal ribosome entry sites (IRES) which allow for translation of protein encoded by RNA which is 3' to the 5' most coding sequence.
- IRES internal ribosome entry sites
- Methods of the invention can be used to construct nucleic acid molecules and cells which contain a wide variety of specific inserts.
- methods of the invention can be used to prepare nucleic acid molecules and cells which contain multiple genes encode specific products. These methods allow for the generation of nucleic acid molecules and organisms which have specific characteristics. For example, as discussed below in Example 18, nucleic acids which contain all of the genes involved in a particular biological pathway can be prepared. Such genes may each be linked to different transcriptional regulatory sequences or one or more copies of the same transcriptional regulatory sequence.
- genes involved in the same or different biological pathways or biological processes may be operably linked to transcriptional regulatory sequences which facilitate transcription in the presence of the same or different inducing agents, under the same or different environmental conditions (e.g., temperature), or in the same or different cell types.
- genes encode polypeptide expression products involved in a pathway or process one or more of these expression products may be expressed as fusion proteins.
- cells can be constructed using methods of the invention which contain inserted nucleic acid segments that encode gene products involved in more than one different biological pathway or biological process.
- One may also use methods of the invention, for example, to modify one or more particular nucleic acid segments in a multi-nucleic acid segment array constructed with a multisite recombination system.
- luxD can be replaced by reacting the vector containing the operon with an appropriate plasmid (e.g. , a pDONR plasmid), such that luxD is substituted with an element comprising ⁇ ttRx-cc ⁇ B-c ⁇ t- ⁇ ttRy to create a vector
- populations of Entry clones with the general configuration of ⁇ ttLx-gene- ⁇ ttLy may be reacted with the product vector, prepared as described above, such that a population of output constructs is generated and for any given construct in the population the segment comprising ⁇ ttRx-cc ⁇ B-c ⁇ t- ⁇ ttRy will have been replaced by another nucleic acid segment flanked by ⁇ ttBx and ⁇ ttBy.
- the ⁇ ttRx-cc ⁇ -c ⁇ t- ⁇ ttRy cassette will have been replaced by a new gene flanked by ⁇ ttBx and ⁇ ttBy.
- nucleic acids segments which encode expression products involved in one or more specific biological processes or pathways may be recombined on supports.
- a first nucleic acid molecule which has a free end on which there is a recombination site and encodes one of three enzymes involved in a biological pathway or process can be attached to a support.
- Nucleic acid molecules of a library having recombination sites on at least one end which are capable of recombining with the nucleic acid molecule attached to the support can then be contacted with the support under conditions which facilitate recombination, leading to the attachment of a second nucleic acid molecule to the first nucleic acid molecule.
- a similar process can be used to attached a third nucleic acid molecule to the free end of the second nucleic acid molecule.
- These resulting nucleic acid products may then be either released from the support prior to assaying for biological activity or such assaying may be performed while the nucleic acid products remain attached the support.
- nucleic acid segments may be cycled on and off the supports described above.
- a second recombination reaction for example, could be used to release the second nucleic acid molecule.
- the invention provides methods for performing recombination between nucleic acid molecules wherein at least one of the nucleic acid molecules is bound to a support.
- the invention further provides methods for identifying nucleic acid molecules involved in the same biological process or pathway by recombining these nucleic acid molecules on supports (e.g. , solid and semi-solid supports).
- supports e.g. , solid and semi-solid supports.
- the invention thus provides methods for screening nucleic acid libraries to identify nucleic acid molecules which encode expression products involved in particular biological processes or pathways, as well as nucleic acid molecules identified by these methods, expressions products produced from the nucleic acid molecules, and products produced by these biological processes or pathways.
- biochemical pathway and “biological pathway” refer to any series of related biochemical reactions that are carried out by an organism or cell. Such pathways may include but are not limited to biosynthetic or biodegradation pathways, or pathways of energy generation or conversion.
- Nucleic acid molecules of the invention can be used for a wide variety of applications. For example, methods of the invention can be used to prepare Destination Vectors which contain all of the structural genes of an operon. As discussed below in Example 18 the lux operon has been reconstructed using nucleic acids encoding the luxCDABE genes obtained from the bioluminescent bacterium Vibrio fischeri.
- nucleic acid molecules of the invention including multiple proteins which are part of the same or different biological pathway or process, can be produced as fusion proteins.
- fusion proteins may contain amino acids which facilitate purification (e.g., 6 His tag),
- target the fusion protein to a particular cellular compartment (e.g., a signal peptide), facilitate solubility (e.g., maltose binding protein), and/or alter the characteristics of the expression product of the cloned gene (e.g., the Fc portion of an antibody molecule, a green flourescent protein (GFP), a yellow fluorescent protein (YFP), or a cyan flourescent protein (CFP)).
- a signal peptide e.g., a signal peptide
- facilitate solubility e.g., maltose binding protein
- alter the characteristics of the expression product of the cloned gene e.g., the Fc portion of an antibody molecule, a green flourescent protein (GFP), a yellow fluorescent protein (YFP), or a cyan flourescent protein (CFP)).
- GFP green flourescent protein
- YFP yellow fluorescent protein
- CFP cyan flourescent protein
- nucleic acid molecule which, upon expression, produce fusion proteins having more than one property, function, or activity.
- a nucleic acid molecule is a molecule which encodes a three component fusion protein comprising a polypeptide of interest, Domain II of Pseudomonas exotoxin, and a polypeptide which promotes binding of the fusion protein to a cell type of interest. Domain II of Pseudomonas exotoxin often confers upon fusion proteins the ability to translocate across cell membranes.
- the expression product could be designed so that it both localizes to a particular cell-type and crosses the cell membrane.
- polypeptide of interest is cytotoxic (e.g., induced apoptosis).
- Nucleic acid molecules which encode proteins similar to those described above are described in Pastan et al, U.S. Patent No. 5,328,984.
- the expression product can be produced in such a manner as to facilitate its export from the cell.
- these expression products can be fusion proteins which contain a signal peptide which results in export of the protein from the cell.
- One application where cell export may be desirable is where the proteins that are to be exported are enzymes which interact with extracellular substrates.
- the invention provides methods for preparing nucleic acid molecules which encode one or more expression products involved in the same or different biological pathway or process, as well as cells which contain these nucleic acid molecules and the resulting products of such biological pathways or processes.
- methods of the invention can be used to construct cells which export multiple proteins involved in the same or different biological processes.
- the invention provides a system for cloning multiple nucleic acid segments in a cell, which export one or more gene products of the expression products of these nucleic acid segments (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.).
- these expression products may perform functions (e.g., catalyze chemical reactions) in extracellular media (e.g., culture media, soils, salt water marshes, etc.).
- nucleic acid molecules When nucleic acid molecules are prepared and/or expressed using methods of the invention, these nucleic acid molecules may encode expression products which are involved in the same or different processes (e.g. , biosynthetic pathways, degradation pathway). As explained below, when one seeks to provide a wide range of functional characteristics to an organism, the nucleic acid molecules may encode expression products which confer relatively unrelated properties upon the organism. Further, nucleic acid molecules can be prepared using methods of the invention which encode all of parts of biosynthetic pathways that lead to desired end products. Further, methods of the invention can be used to generate nucleic acid molecules which encode expression products having unique properties. Thus, the invention also provides methods generating novel end products of biological pathways or processes.
- the invention further provides drug discovery methods and therapeutic agents identified by these methods.
- examples of end products which can be produced by biological pathways or processes reconstituted and/or altered by methods of the invention include chemotherapeutic agents (e.g., antibiotics, antivirals, taxol), carbohydrates, nucleotides, amino acids, lipids, ribosomes, and membrane-bound organdies, as well as novel forms of each.
- chemotherapeutic agents e.g., antibiotics, antivirals, taxol
- carbohydrates e.g., nucleotides, amino acids, lipids, ribosomes, and membrane-bound organdies, as well as novel forms of each.
- the methods of the invention can be used to prepare nucleic acids which confer upon cells the ability to produce a wide variety of natural compounds, as well as modified forms of these compounds.
- Examples of such compounds include those which fall into the following broad classes: anti-bacterial therapeutics, anti- viral therapeutics, anti-parasitic therapeutics, anti-fungal therapeutics, anti-malarial therapeutics, amebicide therapeutics, and anti-neoplastic therapeutics.
- the invention provides methods for producing novel antibiotics, as well as antibiotics produced by methods of the invention.
- an organism which can be produced using methods of the invention is an organism which produces novel antibiotic agents.
- Stassi et al., Proc. Natl. Acad. Sci. USA 95:7305-7309 (1998) describe the production of novel ethyl-substituted erythromycin derivatives produced by genetically engineered cells of Saccharopolyspora erythracea.
- methods of the invention can be used to insert into the cell genetic elements which encode proteins that generate novel antibiotics.
- the invention further includes cells produced by these methods and methods for using such cells to produce antibiotics, as well as antibiotics produced by the methods of the invention.
- Nucleic acid molecules encoding products involved in biosynthetic pathways for numerous therapeutic agents are known in the art. For example, genes and enzymes involved in the biosynthesis of ⁇ -lactam antibiotics are described, for example, in Martin, Appl. Microbiol. Biotechnol. 50(l): ⁇ - ⁇ 5
- the invention includes methods for producing these antibiotics and altered forms of these antibiotics, as well as the antibiotics themselves.
- the invention further provides methods for producing anti-bacterial therapeutics, anti- viral therapeutics, anti-parasitic therapeutics, anti-fungal therapeutics, anti-malarial therapeutics, amebicide therapeutics, and anti-neoplastic therapeutics and altered forms of such agents, as well as the agents themselves.
- anti-bacterial therapeutics include compounds such as penicillins, ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin, cepharadine, cefadoxil, cefaclor, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, imipenem, clavulanate, timentin, sulbactam, erythromycin, neomycin, gentamycin, streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin, quinolones, rifampin, sulfonamides, bacitracin, polymyxin B, vancomycin, doxycycline, methacycline, minocycline
- anti-viral therapeutics examples include acyclovir, idoxuridine, ribavirin, trifluridine, vidirabine, dideoxucytidine, dideoxyinosine, zidovudine and gancyclovir, as well as derivatives and altered forms of each of these compounds.
- anti-parasitic therapeutics include bithionol, diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine, niridazole, oxamniquine (and other quinine derivatives), piperazine citrate, praziquantel, pyrantel pamoate and thiabendazole, as well as derivatives and altered forms of each of these compounds.
- anti-fungal therapeutics include amphotericin B, clotrimazole, econazole nitrate, flucytosine, griseofulvin, ketoconazole and miconazole, as well as derivatives and altered forms of each of these compounds.
- Anti-fungal compounds also include aculeacin A and papulocandin B. (See, e.g. ,
- anti-malarial therapeutics include chloroquine HC1, primaquine phosphate, pyrimethamine, quinine sulfate, and quinacrine HC1, as well as derivatives and altered forms of each of these compounds.
- amebicide therapeutics include dehydroemetine dihydrochloride, iodoquinol, andparamomycin sulfate, as well as derivatives and altered forms of each of these compounds.
- anti-neoplastic therapeutics include aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubicin, doxorubicin, taxol, etoposide, fluorouracil, interferon- ⁇ , lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HC1, thioguanine, vinblastine sulfate and vincristine sulfate, as well as derivatives and altered forms of each of these compounds.
- Additional anti-microbial agents include peptides.
- anti-microbial peptides are disclosed in Hancock et al. , U.S . Patent 6,040,435 and Hancock et al, Proc. Natl. Acad. Sci. USA 97:8856-8861 (2000).
- Nucleic acid molecules can also be prepared using the methods of the invention which encode more than one subunit of a multi-protein complex. Examples of such multi-protein complexes include splicesomes, ribosomes, the human 26S proteasome, and yeast RNA polymerase III. (See, e.g., Saito et al, Gene 05 :241-250 (1997); Flores et al, Proc. Natl. Acad. Sci. USA 95(7 >:7815-7820 (1999).)
- Methods of the invention can also be used for the partial synthesis of non-naturally occurring products, as well as variants of these products (e.g., novel variants).
- microorganisms which express enzymes which catalyze particular reactions can be supplied with precursors which these organisms do not normally produce.
- novel compounds may be produced.
- “Feeding" processes of this type have been used in the past to produce novel antibiotics. In one aspect, feeding of this type is used in combination with microorganisms which express enzymes encoded by combinatorial libraries described above.
- Methods of the invention can be used to either (1) introduce a new pathway into a cells or (2) alter an existing cellular pathway so that, for example, one or more additional catalytic steps (e.g. , two, three, four, five, seven, ten, etc.) occur during product synthesis.
- one or more additional catalytic steps e.g. , two, three, four, five, seven, ten, etc.
- One example of such an application of methods of the invention involves the modification of a protein which is naturally produced by a cell.
- genes encoding one or more catalytic steps which alter the protein are introduced into the cell.
- nucleic acids which encode enzymes involved in ADP-ribosylation, glycosylation, sialylation, acetylation, ubiquination, serine to D-alanine conversion, biotinylation, acylation, amidation, formylation, carboxylation, GPI anchor formation, hydroxylation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation can be inserted into the cell.
- Post-translational modifications of proteins are discussed in PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,
- Methods of the invention can be used, for example, to produce cells which contain nucleic acid molecules which encode proteins involved in signaling pathways. Further, these cells may be used to screen agents which modulate cell signaling. For example, cells may be produced using methods of the invention which express all of the components necessary for responding to tumor necrosis factors (TNFs). These cells can then be used to screen agents which either induce TNF mediated responses (TNF agonists) or block TNF mediated responses (TNF antagonists). Thus, included within the scope of the invention are methods for producing cells which can be used to screen for agonists and antagonists of cellular ligands, as well as cells produced by such methods. Further included within the scope of the invention are methods for using cells of the invention to identify agonists and antagonists of cellular ligands and agonists and antagonists identified by methods of the invention.
- TNFs tumor necrosis factors
- methods of the invention can also be used to generate nucleic acids and cells which produce nutrients such as carbohydrates and amino acids.
- Carbohydrates and amino acids, as well as other carbon sources can be used for a number of purposes.
- carbohydrates and amino acids to prepare culture medium components for growing microorganisms, mammalian cells, and plant cells. Further, these compounds can be added to food products for both humans and liverstock.
- One specific example of a use of carbohydrates and amino acids is in the preparation of nutritional formula for infants. (See, e.g. , Highman et al. , U.S. Patent No. 6,120,814.)
- the invention further provides food products (e.g., infant formula) made using carbon sources produced using methods of the invention.
- Carbon sources which can be produced using cells prepared using methods of the invention include carbohydrates (e.g., glucose, fructose, lactose, molasses, cellulose hydrolyzates, crude sugar hydrolyzates, and starch hydrolyzates), organic acids (e.g., pyruvic acid, acetic acid, fumaric acid, malic acid, and lactic acid), alcohols (glycerol, 1,3,-propanediol, and ethanol), lipids, fatty acids, nucleotides, nucleosides, and amino acids. (See, e.g., Skraly et al, Appl. Environ Microbiol. 54:98-105 (1998).)
- carbohydrates e.g., glucose, fructose, lactose, molasses, cellulose hydrolyzates, crude sugar hydrolyzates, and starch hydrolyzates
- organic acids e.g., pyruvic acid, acetic acid, fumaric acid, malic acid,
- an organism which can be produced using methods of the invention is an organism which has acquired the ability to produce ethanol.
- methods of the invention can be used to insert into cell genetic elements which encode proteins involved in the production of ethanol.
- the invention further includes cells produced by these methods and methods for using such cells to produce ethanol.
- Another example of an organism which can be produced using methods of the invention is an organism which has acquired the ability to produce either poly(3-hydroxyalkanoates) or increased amounts of poly(3-hydroxyalkanoates).
- Poly(3-hydroxyalkanoates) are compounds which, on extraction from cells, have plastic like properties. (See, e.g., Madison et al, Microbiol. Molec. Biol. Rev. 55:21-53 (1999).)
- methods of the invention can be used to insert into cell genetic elements which encode proteins involved in the production of poly(3-hydroxyalkanoates).
- the invention further includes cells produced by these methods and methods for using such cells to produce poly(3-hydroxyalkanoates), poly(3-hydroxyalkanoates) derivatives, and compounds formed from poly(3-hydroxyalkanoates).
- Amino acids which can be produced using cells prepared using methods of the invention include phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, glutamine, asparagine, arginine, lysine, histidine, aspartic acid, glutamic acid, alanine, proline, serine, threonine, methionine, cysteine, and glycine.
- methods of the invention can also be used to engineer cells so that they either overproduce or underproduce products of the cells normal metabolism.
- Donnelly et al U.S. Patent No. 5,770,435 described a mutant strain of E. coli which produce increased amounts of succinic acid.
- Methods of the invention can be used, for example, to construct nucleic acid molecules which encode enzymes in the succinic acid biosynthetic pathway. Further, the expression of one or more of these enzymes can be regulated at the transcriptional level.
- the introduction of these nucleic acid molecules into the above described E. coli cells will effectively result in an amplification of one or more genes in the succinic acid biosynthetic pathway.
- one or more of these genes can be operably linked to an inducible promoter (e.g. , the lacl promoter) so that increased succinic acid occurs only in the presence of the inducing signal (e.g., IPTG).
- an inducible promoter e.g. , the lacl
- Methods of the invention can also be used to generate nucleic acids and cells which produce components and precursors that can be used in manufacturing processes.
- components include plastics, plastic-like compounds (e.g., polyketides), soaps, fertilizers, papers, synthetic rubber, dyes, inks, etc.
- the invention further includes components and precursors produced by methods and cells of the invention.
- nucleic acid molecules prepared by the methods of the invention can also be used to down regulate expression of, for example, one or more endogenous genes.
- nucleic acid inserts prepared by methods of the invention are transcribed to produce antisense RNA.
- nucleic acid molecules which encode antisense RNAs may be operably linked to a regulatable promoter.
- the invention further includes methods for producing cells which either overproduce or underproduce products of the cells normal metabolism, as well as cells produced by these methods.
- nucleic acid molecules prepared by methods of the invention can be used to alter the physical characteristics of an organism so that the organism has particular characteristics.
- a cell which lacks specific enzymes required to produce either recombinant or native proteins having particular glycosylation patterns can be introduced into the cell using the vectors of the invention. Glycosylation patterns of proteins has been found to be, to some extent, cell-type and species specific. (See, e.g. , Jarvis et al, Curr. Opin. Biotechnol.
- the invention provides methods for producing cells which exhibit altered glycosylation pathways, as well as cells produced by these methods and glycosylated compounds produced by these cells. This process is generally termed "glycosylation engineering.”
- bacterial cells which do not glycosylate proteins may be modified using methods of the invention to produce enzymes which glycosylate proteins.
- enzymes include N-acetylglucosaminlytransferases III and V, ⁇ l ,4-galactosyltransferase, ⁇ 2,6-sialyltransferase, ⁇ 2,3-sialyltransferase, ⁇ l ,3-fucosyltransferase III and VI, and ⁇ l ,2-mannosyltransferase.
- the invention provides methods for producing cells which exhibit altered metabolic properties leading to increased production of compounds synthesized by these cells, as well as cells produced by these methods and products produced by these cells.
- One example of such methods result in the production of cells which produce increased quantities of precursors for biological pathways. This process is referred to herein as metabolic channeling or funneling.
- metabolic channeling or funneling For example, when one seeks to produce a cell which produces increased amounts of serine, nucleic acid molecules which encode enzymes of pathways which lead to the production of 3-phosphoglycerate can be inserted into the cell.
- nucleic acid molecules which encode enzymes involved in the conversion of 3-phosphoglycerate to serine can also be inserted into the cell.
- Parameters useful for consideration when engineering cells which contain increased intracellular concentrations of precursor pools an compounds include the rate limiting set in the particular pathway and pathway fluxes. (See, e.g., Kholodenko et al, Biotechnol. Bioeng. 59:239-247 (1997).)
- Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides are produced in many types of organisms, including fungi and numerous bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures and polyketides encompasses numerous compounds with diverse activities. (See, e.g., PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos.
- PKSs Polyketide synthases
- PKSs generally catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA.in a biosynthetic process that closely parallels fatty acid biosynthesis.
- polyketides include callystatin A, ansatrienin A, actinorhodin, rapamycin, methymycin, and pikromycin.
- the invention provides methods for preparing nucleic acid molecules which encode one or more PKSs, as well as cells which contain these nucleic acid molecules and the resulting polyketide products.
- the invention further provides methods for generating novel PKSs using combinatorial libraries and products produced by these novel PKSs (e.g., novel macrolide antibiotics), as well methods for producing these novel PKS products.
- Methods of the invention can also be used to construct strains of microorganisms which are useful for decreasing the toxicity of various agents.
- agents include petroleum-based pollutants (e.g., chlorinated and non-chlorinated aliphatic compounds (e.g., C 5 -C 36 ), chlorinated and non-chlorinated aromatic compounds (e.g.
- an organism which can be produced using methods of the invention is an organism which degrades toluene.
- Panke et al, App Environ. Microbiol. 54:748-751 (1998) describe strains of Pseudomonas putida which converts toluene, as well as several toluene derivatives, to benzoates.
- methods of the invention can be used to insert into cell genetic elements which encode proteins that convert toluene, as well as derivatives thereof, to less toxic compounds.
- the invention further includes cells produced by these methods and methods for using such cells to convert toluene, as well as several toluene derivatives, to less toxic compounds.
- Methods of the invention can also be used to prepare organism suitable for the detoxifying non-petroleum agents such as heavy metal ions (e.g. , mercury, copper, cadmium, silver, gold, tellurite, selenite, and uranium).
- non-petroleum agents such as heavy metal ions (e.g. , mercury, copper, cadmium, silver, gold, tellurite, selenite, and uranium).
- heavy metal ions e.g. , mercury, copper, cadmium, silver, gold, tellurite, selenite, and uranium.
- Methods by which mercury, for example, can be detoxified include reduction of mercury ions to generate metallic mercury and through volatilization.
- Genes involved in the detoxification by bacterial are described in Miller, "Bacterial Detoxification of
- Microorganisms which are capable of detoxifying agents are described, for example, in Perriello, U.S. Patent No. 6,110,372.
- Microorganisms suitable for bioremediation applications include those of the Pseudomonadaceae family, the Actinomycetes family, the Micrococcaceae family, the Vibrionaceae family, the Rhizobiaceae family, the Cytophagaceae family, and the Corynebacterium family.
- organisms suitable for use after modification using the methods of the invention for bioremediation applications include Pseudomonas rubrisubalbicans, Pseudomonas aeruginosa, Variovorax paradoxus, Nocardia asteroides, Deinococcus radiodurans, Nocardia restricta, Chryseobacterium indologenes, Comamonas acidovorans, Acidovoraxdelafieldii, Rhodococcus rhodochrous, Rhodococcus erytlropolis, Aureobacterium esteroaromaticum, Aureobacterium saperdae, Micrococcusvarians, Micrococcus kristinae, Aeromonas caviae, Stenotrophomonas maltophilia, Sphingobacterium thalpophilum, Clavibacter michiganense, Alcaligenes xylosoxydans,
- Organisms suitable for bioremediation further include plants.
- Meagher et al U.S. Patent No. 5,965,796, for example, describes transgenic plants which express a metal ion resistance protein and reduce metal ions such as those of copper, mercury, gold, cadmium, lead and silver.
- genes encoding phytochelatins can be introduced into plants to increase phytochelatin synthesis.
- Phytochelatins are glutathione derivatives which detoxify metal ions through sequestration. Genes from a number of plant species involved in phytochelatin synthesis are discussed in Corbett, "Phytochelatin Biosynthesis and Function in Heavy-Metal Detoxification ", Curr. Opin. Plant Biol. 3(3):2 ⁇ ⁇ -2 ⁇ 6 (2000).
- Specific plants suitable for bioremediation applications after modification by methods of the invention include Lepidium sativum, Brassicajuncea, Brassica oleracea, Brassica rapa, Acena sativa, Triticum aestivum, Helianthus annuus, Colonial bentgrass, Kentucky bluegrass, perennial ryegrass, creeping bentgrass, Bermudagrass, Buffalograss, centipedegrass, switch grass, Japanese lawngrass, coastal panicgrass, spinach, sorghum, tobacco and corn.
- Methods for generating transgenic plants are known in the art and, as noted above, are described, for example, in Meagher et al, U.S. Patent No. 5,965,796.
- Methods of the invention can also be used to prepare organisms which have diverse characteristics and contain a considerable number of inserted genes.
- methods of the invention can be used to insert an almost unlimited number of nucleic acid segments into cells.
- the invention provides methods for producing cells which express pesticidal proteins (e.g., pesticidal proteins of Bacillus thurginiensis).
- pesticidal proteins e.g., pesticidal proteins of Bacillus thurginiensis
- methods of the invention can be used to insert into cell genetic elements which encode pesticidal proteins.
- the invention further includes cells produced by these methods and methods for using such cells to produce pesticidal proteins.
- the invention further includes methods for using cells (e.g. , bacterial or plant cells) and pesticidal proteins produced by methods of the invention to control insect populations.
- cells produced by methods of the invention and used in methods of the invention will be plant cells.
- methods of the invention may be used to prepare nucleic acid molecules which contain one or more ORFs and/or nucleic acid segments which encode one or more non-protein expression products (e.g., functional RNAs such as tRNAs or ribozymes).
- the number of ORFs and/or nucleic acid segments which encode one or more non-protein expression products will generally range between about 1 and about 300 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
- Nucleic acid molecules which contain one or more ORFs and/or nucleic acid segments which encode one or more non-protein expression products will be especially useful for altering organisms to have specified characteristics such as those described above.
- the size of nucleic acid molecules of the invention will vary considerably in size but, in general, will range between from about 0.5 kb to about 300 kb (e.g., about 0.5 kb, about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 7 kb, about 10 kb, about 12 kb, about 15 kb, about 20 kb, about
- the invention further provides methods for introducing nucleic acid molecules of the invention into animals (e.g., humans) and animal cells (e.g., human cells), as part of a gene therapy protocol.
- Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid molecule.
- nucleic acid molecules of the invention will encoded one or more proteins which mediates at least one therapeutic effect.
- the invention provide nucleic acid molecules and methods for use in gene therapy.
- Nucleic acid molecules of the invention can be used to prepare gene therapy vectors designed to replace genes which reside in the genome of a cell, to delete such genes, or to insert a heterologous gene or groups of genes.
- nucleic acid molecules of the invention function to delete or replace a gene or genes, the gene or genes being deleted or replaced may lead to the expression of either a "normal" phenotype or an aberrant phenotype.
- an aberrant phenotype is the disease cystic fibrosis.
- the gene therapy vectors may be either stably maintained (e.g. , integrate into cellular nucleic acid by homologous recombination) or non-stably maintained in cells.
- nucleic acid molecules of the invention may be used to suppress
- nucleic acid molecule of the invention designed to suppress an abnormal phenotype would be where an expression product of the nucleic acid molecule has dominant/negative activity.
- nucleic acid molecule of the invention designed to supplement a normal phenotype would be where introduction of the nucleic acid molecule effectively results in the amplification of a gene resident in the cell.
- nucleic acid molecules of the invention may be used to insert into cells nucleic acid segments which encode expression products involved in each step of particular biological pathways (e.g., biosynthesis of amino acids such as lysine, threonine, etc.) or expression products involved in one or a few steps of such pathways.
- These nucleic acid molecules can be designed to, in effect, amplify genes encoding expression products in such pathways, insert genes into cells which encode expression products involved in pathways not normally found in the cells, or to replace one or more genes involved one or more steps of particular biological pathways in cells.
- gene therapy vectors of the invention may contain nucleic acid which results in the production one or more products (e.g., one, two, three, four, five, eight, ten, fifteen, etc.).
- the invention provides gene therapy vectors which express one or more expression products (e.g., one or more fusion proteins), methods for producing such vectors, methods for performing gene therapy using vectors of the invention, expression products of such vector (e.g., encoded RNA and/or proteins), and host cells which contain vectors of the invention.
- one or more expression products e.g., one or more fusion proteins
- methods for producing such vectors methods for performing gene therapy using vectors of the invention, expression products of such vector (e.g., encoded RNA and/or proteins), and host cells which contain vectors of the invention.
- nucleic acid molecules of the invention may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
- nucleic acid molecules of the invention are directly administered in vivo, where they are expressed to produce one or more expression products.
- This can be accomplished by any of numerous methods known in the art, such as by constructing an expression vector and administering it so that they become intracellular (e.g., by infection using defective or attenuated retroviral vectors or other viral vectors (see U.S. Patent No. 4,980,286), by direct injection of naked DNA, by use of microparticle bombardment (e.g.
- a gene gun Biolistic, Dupont
- Biolistic, Dupont by coating with lipids or cell- surface receptors or transfecting agents, encapsulation in Hposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (.see, e.g., Wu and Wu, 1987, J. Biol. Chem. 252:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.).
- nucleic acid molecules of the invention can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g. , PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al. ); WO92/20316 dated November 26, 1992 (Findeis et al. ); WO93/14188 dated July 22, 1993 (Clarke et al), WO 93/20221 dated October 14, 1993 (Young)).
- a specific receptor see, e.g. , PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al. ); WO92/20316 dated November 26, 1992 (Findeis et al. ); WO93/14188 dated July 22, 1993 (Clarke et al), WO
- nucleic acid molecules of the invention can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstraet ⁇ /., 1989, Nature 342:435-438).
- Example of such nucleic acid construct suitable for such an application are shown in Figures 21 C and 22B.
- viral vectors that contains nucleic acid sequences encoding an antibody or other antigen-binding protein of the invention are used.
- a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol.217:581 -599). These retroviral vectors have been used to delete retro viral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
- the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
- retroviral vectors More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the marl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
- Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al. , 1994, J. Clin. Invest.93 :644-651 ; Kiem et al. , 1994, Blood 83:1467- 1473 ; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,
- Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia and the use of such vectors are included within the scope of the invention. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al, 1994, Human Gene
- adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys.
- adenovirus vectors are used.
- Adeno-associated virus AAV
- Herpes viruses as well as vectors prepared from these viruses have also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No. 5,436,146; Wagstaff et ⁇ /., Gene Ther. 5:1566-70 (1998)).
- Herpes viral vectors are particularly useful for applications where gene expression is desired in nerve cells.
- Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
- the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
- the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
- introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
- Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g. , Loeffler and Behr, 1993, Meth. Enzymol.217:599-618; Cohen etal, 1993, Meth. Enzymol.217:618-644; Cline,
- the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and, optionally, heritable and expressible by its cell progeny.
- the resulting recombinant cells can be delivered to a patient by various methods known in the art.
- Recombinant blood cells e.g. , hematopoietic stem or progenitor cells
- the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
- Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.).
- the cell used for gene therapy is autologous to the patient.
- nucleic acid sequences encoding an antibody or other antigen-binding protein are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
- stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see, e.g., PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71 :973-985; Rheinwald, 1980, Meth. Cell Bio. 21 A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
- nucleic acid molecules to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid molecules are controllable by controlling the presence or absence of the appropriate inducer of transcription.
- the nucleic acid molecules of the invention can also be used to produce transgenic organisms (e.g., animals and plants).
- Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates (e.g. , baboons, monkeys, and chimpanzees) may be used to generate transgenic animals.
- plants of any species including but not limited to Lepidium sativum, Brassica juncea, Brassica oleracea, Brassica rapa, Acena sativa, Triticum aestivum, Helianthus annuus, Colonial bentgrass, Kentucky bluegrass, perennial ryegrass, creeping bentgrass, Bermudagrass, Buffalograss, centipedegrass, switch grass, Japanese lawngrass, coastal panicgrass, spinach, sorghum, tobacco and corn, may be used to generate transgenic plants.
- Any technique known in the art may be used to introduce nucleic acid molecules of the invention into organisms to produce the founder lines of transgenic organisms.
- Such techniques include, but are not limited to, pronuclear microinjection (Patersonet ⁇ /.,-4/?p/. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al, Biotechnology (NY) 77:1263-1270 (1993); Wright et al, Biotechnology (NY) 9:830-834 (1991); and Hoppe etal, U.S. Pat. No.4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al. , Proc. Natl. Acad.
- transgenic clones containing nucleic acid molecules of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al, Nature 550:64-66 (1996); Wilmut et al, Nature 555:810-813 (1997)), each of which is herein incorporated by reference in its entirety).
- the present invention provides for transgenic organisms that carry nucleic acid molecules of the invention in all their cells, as well as organisms which carry these nucleic acid molecules, but not all their cells, i.e., mosaic organisms or chimeric.
- the nucleic acid molecules of the invention may be integrated as a single copy or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
- the nucleic acid molecules of the invention may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al, Proc. Natl. Acad. Sci. USA 59:6232-6236 (1992)).
- the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
- nucleic acid molecules of the invention When it is desired that nucleic acid molecules of the invention be integrated into the chromosomal site of the endogenous gene, this will normally be done by gene targeting. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. Nucleic acid molecules of the invention may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al, Science 255:103-106 (1994)).
- transgenic organisms Once transgenic organisms have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze organism tissues to verify that integration of nucleic acid molecules of the invention has taken place.
- the level of mRNA expression of nucleic acid molecules of the invention in the tissues of the transgenic organisms may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the organism, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of tissue may which express nucleic acid molecules of the invention also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the expression product of these nucleic acid molecules.
- founder organisms may be bred, inbred, outbred, or crossbred to produce colonies of the particular organism.
- breeding strategies include, but are not limited to: outbreeding of founder organisms with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenic organisms that express nucleic acid molecules of the invention at higher levels because of the effects of additive expression of each copy of nucleic acid molecules of the invention; crossing of heterozygous transgenic organisms to produce organisms homozygous for a given integration site in order to both augment expression and eliminate the need for screening of organisms by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the nucleic acid molecules of the invention on a distinct background that is appropriate for an experimental model of interest.
- Transgenic and "knock-out" organisms of the invention have uses which include, but are not limited to, model systems (e.g. , animal model systems) useful in elaborating the biological function of expression products of nucleic acid molecules of the invention, studying conditions and or disorders associated with aberrant expression of expression products of nucleic acid molecules of the invention, and in screening for compounds effective in ameliorating such conditions and/or disorders.
- model systems e.g. , animal model systems
- tissue-specific transcriptional regulatory sequences e.g., tissue-specific promoters
- tissue-specific promoters e.g., tissue-specific promoters
- tissue-specific promoters can be used to facilitate production of these expression products in desired tissues.
- tissue-specific promoters are known in the art. Further, methods for identifying tissue-specific transcriptional regulatory sequences are described elsewhere herein.
- the invention also relates to host cells comprising one or more of the nucleic acid molecules or vectors of the invention (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), particularly those nucleic acid molecules and vectors described in detail herein.
- Representative host cells that may be used according to this aspect of the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells.
- Preferred bacterial host cells include Escherichia spp. cells (particularly E. coli cells and most particularly E. coli strains DH10B, Stbl2, DH5 ⁇ , DB3, DB3.1 (preferably
- P. aeruginosa cells and Salmonella spp. cells (particularly S. typhimurium and S. typhi cells).
- Preferred animal host cells include insect cells (most particularly Drosophila melanogaster cells, Spodopterafrugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells), nematode cells (particularly C. elegans cells), avian cells, amphibian cells
- yeast host cells include Saccharomyces cerevisiae cells and Pichia pastor is cells. These and other suitable host cells are available commercially, for example, from Invitrogen Corp., Life Technologies Division (Rockville, Maryland), American
- nucleic acid molecules and/or vectors of the invention may be introduced into host cells using well known techniques of infection, transduction, electroporation, transfection, and transformation.
- the nucleic acid molecules and/or vectors of the invention may be introduced alone or in conjunction with other the nucleic acid molecules and/or vectors and/or proteins, peptides or RNAs.
- nucleic acid molecules and/or vectors of the invention may be introduced into host cells as a precipitate, such as a calcium phosphate precipitate, or in a complex with a lipid. Electroporation also may be used to introduce the nucleic acid molecules and/or vectors of the invention into a host. Likewise, such molecules may be introduced into chemically competent cells such as E. coli. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells. Thus nucleic acid molecules of the invention may contain and/or encode one or more packaging signal (e. g. , viral packaging signals which direct the packaging of viral nucleic acid molecules).
- packaging signal e. g. , viral packaging signals which direct the packaging of viral nucleic acid molecules.
- Polymerases for use in the invention include but are not limited to polymerases (DNA and RNA polymerases), and reverse transcriptases.
- DNA polymerases include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENTTM) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENTTM DNA polymerase,
- Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp KOD2 (KOD) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYMETM) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA polymerase
- RNA polymerases such as T3, T5, T7 and SP6 and mutants, variants and derivatives thereof may also be used in accordance with the invention.
- the nucleic acid polymerases used in the present invention may be mesophilic or thermophilic, and are preferably thermophilic.
- Preferred mesophilic DNA polymerases include Pol I family of DNA polymerases (and their respective Klenow fragments) any of which may be isolated from organism such as E. coli, H. influenzae, D. radiodurans, H. pylori, C. aurantiacus, R. prowazekii, T. pallidum, Synechocystis sp., B. subtilis, L. lactis, S. pneumoniae, M. tuberculosis, M. leprae, M.
- thermostable DNA polymerases that may be used in the methods and compositions of the invention include Taq, Tne, Tma, Pfu, KOD, Tfl, Tth, Stoffel fragment, VENTTM and DEEPVENTTM DNA polymerases, and mutants, variants and derivatives thereof (U.S. Patent No. 5,436,149; U.S. Patent 4,889,818; U.S. Patent 4,965,188; U.S.
- Reverse transcriptases for use in this invention include any enzyme having reverse transcriptase activity.
- Such enzymes include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R.K., et al, Science 239:487-491 (1988); U.S. Patent Nos. 4,889,818 and 4,965,188), Tne DNA polymerase (WO 96/ 10640 and WO 97/09451 ), Tma DNA polymerase (U. S. Patent No.
- Preferred enzymes for use in the invention include those that have reduced, substantially reduced or eliminated RNase H activity.
- an enzyme “substantially reduced in RNase H activity” is meant that the enzyme has less than about 20%, more preferably less than about 15%, 10%) or 5%, and most preferably less than about 2%, of the RNase H activity of the corresponding wild-type or RNase H + enzyme such as wild-type
- M-MLV Moloney Murine Leukemia Virus
- AMV Avian Myeloblastosis Virus
- RSV Rous Sarcoma Virus reverse transcriptases.
- the RNase H activity of any enzyme may be determined by a variety of assays, such as those described, for example, in U.S. Patent No. 5,244,797, in Kotewicz, M.L., et al, Nucl. Acids Res. 16:265 (1988) and in Gerard, G.F., et al, FOCUS 14(5):91 (1992), the disclosures of all of which are fully incorporated herein by reference.
- polypeptides for use in the invention include, but are not limited to, M-MLV H " reverse transcriptase, RSV H “ reverse transcriptase, AMV H “ reverse transcriptase, RAV (rous-associated virus) H “ reverse transcriptase, MAV (myeloblastosis-associated virus) H “ reverse transcriptase and HIV H “ reverse transcriptase.
- M-MLV H " reverse transcriptase reverse transcriptase
- RSV H reverse transcriptase
- AMV H reverse transcriptase
- RAV rous-associated virus
- MAV myeloblastosis-associated virus
- HIV H reverse transcriptase
- any enzyme capable of producing a DNA molecule from a ribonucleic acid molecule may be equivalently used in the compositions, methods and kits of the invention.
- the enzymes having polymerase activity for use in the invention may be obtained commercially, for example from Invitrogen Corp., Life Technologies Division (Rockville, Maryland), Perkin-Elmer (Branchburg, New Jersey), New England BioLabs (Beverly, Massachusetts) or Boehringer Mannheim
- Enzymes having reverse transcriptase activity for use in the invention may be obtained commercially, for example, from Invitrogen Corp., Life Technologies Division (Rockville, Maryland), Pharmacia (Piscataway, New Jersey), Sigma (Saint Louis, Missouri) or Boehringer Mannheim Biochemicals (Indianapolis, Indiana).
- polymerases or reverse transcriptases having polymerase activity may be isolated from their natural viral or bacterial sources according to standard procedures for isolating and purifying natural proteins that are well-known to one of ordinary skill in the art (see, e.g. , Houts, G.E., et al, J. Virol.29:517 (1979)).
- polymerases/reverse transcriptases may be prepared by recombinant DNA techniques that are familiar to one of ordinary skill in the art (see, e.g. , Kotewicz, M.L., et al, Nucl. Acids Res. 16:265 (1988); U.S. Patent No. 5,244,797; WO 98/47912; Soltis, D.A., and Skalka, A.M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)).
- enzymes having polymerase activity and reverse transcriptase activity may include any of those described in the present application.
- Supports for use in accordance with the invention may be any support or matrix suitable for attaching nucleic acid molecules comprising one or more recombination sites or portions thereof. Such molecules may be added or bound
- Supports of the invention may comprise nitrocellulose, diazocellulose, glass, polystyrene (including microtitre plates), polyvinylchloride, polypropylene, polyethylene, polyvinylidenedifluoride (PVDF), dextran, Sepharose, agar, starch and nylon.
- Supports of the invention may be in any form or configuration including beads, filters, membranes, sheets, frits, plugs, columns and the like.
- Solid supports may also include multi-well tubes (such as microtitre plates) such as 12-well plates, 24-well plates, 48-well plates, 96-well plates, and 384-well plates.
- Preferred beads are made of glass, latex or a magnetic material (magnetic, paramagnetic or superparamagnetic beads).
- methods of the invention may be used to prepare arrays of proteins or nucleic acid molecules (RNA or DNA) or arrays of other molecules, compounds, and/or substances.
- arrays may be formed on microplates, glass slides or standard blotting membranes and may be referred to as microarrays or gene-chips depending on the format and design of the array. Uses for such arrays include gene discovery, gene expression profiling, genotyping (SNP analysis, pharmacogenomics, toxicogenetics), and the preparation of nanotechnology devices.
- nucleic acid molecules comprising one or more recombination sites or portions thereof can be added to one or more supports (or can be added in arrays on such supports) and nucleic acids, proteins or other molecules and/or compounds can be added to such supports through recombination methods of the invention.
- Conjugation of nucleic acids to a molecule of interest are known in the art and thus one of ordinary skill can produce molecules and/or compounds comprising recombination sites (or portions thereof) for attachment to supports (in array format or otherwise) according to the invention.
- any conceivable support may be employed in the invention.
- the support may be biological, non-biological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc.
- the support may have any convenient shape, such as a disc, square, sphere, circle, etc.
- the support is preferably flat but may take on a variety of alternative surface configurations.
- the support may contain raised or depressed regions which may be used for synthesis or other reactions.
- the support and its surface preferably form a rigid support on which to carry out the reactions described herein.
- the support and its surface are also chosen to provide appropriate light-absorbing characteristics.
- the support may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , S1N 4 , modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof.
- gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof.
- Other support materials will be readily apparent to those of skill in the art upon review of this disclosure.
- the support is flat glass or single-crystal silicon.
- these nucleic acid molecules will have recombination sites at one or more (e.g., one, two, three or four) of their termini.
- one nucleic acid molecule will be attached directly to the support, or to a specific section of the support, and one or more additional nucleic acid molecules will be indirectly attached to the support via attachment to the nucleic acid molecule which is attached directly to the support.
- the nucleic acid molecule which is attached directly to the support provides a site of nucleation around which a nucleic acid array may be constructed.
- the invention further provides methods for linking supports to each other and for linking molecules bound to the same support together.
- a recombination site designated RS 6 can be positioned at the end of the RS 5 site on the A/B composition shown attached to the support in the lower portion of the figure.
- an identical composition may also be attached to another part of the same or different support. Recombination between the RS 6 sites can then be used to connect the two compositions, thereby forming either a linkage between two compositions attached to the same support or two compositions attached to the different support.
- the invention thus provides methods for cross-linking compounds attached to the same support by linking one or more compositions bound to the support using recombination sites.
- the invention also provides methods for cross-linking separate supports by linking one or more compositions bound to these supports suing recombination sites.
- the invention provides supports containing nucleic acid molecules which are produced by methods of the invention.
- the nucleic acid molecules of these supports will contain at least one recombination site. In some embodiments, this recombination site will have undergone recombination prior to attachment of the nucleic acid molecule to the support.
- These bound nucleic acid molecules are useful, for example, for identifying other nucleic acid molecules (e.g., nucleic acid molecules which hybridize to the bound nucleic acid molecules under stringent hybridization conditions) and proteins which have binding affinity for the bound nucleic acid molecules.
- Expression products may also be produced from these bound nucleic acid molecules while the nucleic acid molecules remain bound to the support.
- compositions and methods of the invention can be used to identify expression products and products produced by these expression products.
- nucleic acid molecules bound to supports will undergo recombination after attachment of the nucleic acid molecule to the support.
- these bound nucleic acid molecules may thus be used to identify nucleic acid molecules which encode expression products involved in one or a specified number of biological processes or pathways.
- nucleic acid molecules attached to supports may be released from these supports.
- Methods for releasing nucleic acid molecules include restriction digestion, recombination, and altering conditions (e.g., temperature, salt concentrations, etc.) to induce the dissociation of nucleic acid molecules which have hybridized to bound nucleic acid molecules.
- methods of the invention include the use of supports to which nucleic acid molecules have been bound for the isolation of nucleic acid molecules.
- the invention provides methods for screening nucleic acid libraries to identifying nucleic acid molecules which encode expression products involved in the same biological processes or pathways.
- such methods involve (1) attaching a nucleic acid molecule comprising at least one recombination site to a support, (2) contact the bound nucleic acid molecule with a library of nucleic acid molecules, wherein individual nucleic acid molecules of the library comprise at least one recombination site, under conditions which facilitate recombination between the bound nucleic acid molecule and nucleic acid molecules of the library, and (3) screening for either expression products of the nucleic acid molecule formed by recombination or products produced by the expression products of these nucleic acid molecules.
- compositions which can be formed by binding nucleic acid molecules to supports are “gene chips,” often referred to in the art as “DNA microarrays” or “genome chips” (see U.S. Patent Nos. 5,412,087 and 5,889,165, and PCT Publication Nos. WO 97/02357, WO 97/43450, WO 98/20967, WO 99/05574, WO 99/05591, and WO 99/40105, the disclosures of which are incorporated by reference herein in their entireties).
- these gene chips may contain two- and three-dimensional nucleic acid arrays described herein.
- nucleic acid arrays of the invention means that molecules or compounds which bind to particular nucleotide sequences can be attached to the arrays.
- components such as proteins and other nucleic acids can be attached to specific locations/positions in nucleic acid arrays of the invention.
- the invention provides affinity purification methods comprising (1) providing a support to which nucleic acid molecules comprising at least one recombination site are bound, (2) attaching one or more additional nucleic acid molecules to the support using recombination reactions,
- the present invention may be used in combination with any method involving the synthesis of nucleic acid molecules, such as DNA (including cDNA) and RNA molecules.
- nucleic acid molecules such as DNA (including cDNA) and RNA molecules.
- methods include, but are not limited to, nucleic acid synthesis methods, nucleic acid amplification methods and nucleic acid sequencing methods.
- Such methods may be used to prepare molecules (e.g. , starting molecules) used in the invention or to further manipulate molecules or vectors produced by the invention.
- Nucleic acid synthesis methods may comprise one or more steps (e.g., two, three, four, five, seven, ten, twelve, fifteen, etc.).
- the invention provides a method for synthesizing a nucleic acid molecule comprising (a) mixing a nucleic acid template (e.g., a nucleic acid molecules or vectors of the invention) with one or more primers (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) and one or more enzymes (e.g., two, three, four, five, seven, etc.) having polymerase or reverse transcriptase activity to form a mixture; and (b) incubating the mixture under conditions sufficient to make a first nucleic acid molecule complementary to all or a portion of the template.
- a nucleic acid template e.g., a nucleic acid molecules or vectors of the invention
- primers e.g., two, three, four, five, seven
- the nucleic acid template may be a DNA molecule such as a cDNA molecule or library, or an R A molecule such as a mRNA molecule.
- Conditions sufficient to allow synthesis such as pH, temperature, ionic strength, and incubation times may be optimized by those skilled in the art. If desired, recombination sites may be added to such synthesized molecules during or after the synthesis process (see, e.g., U.S. Patent Application No. 09/177,387 filed 10/23/98 based on U.S. provisional patent application no. 60/065,930 filed October 24, 1997).
- the target or template nucleic acid molecules or libraries may be prepared from nucleic acid molecules obtained from natural sources, such as a variety of cells, tissues, organs or organisms.
- Cells that may be used as sources of nucleic acid molecules may be prokaryotic (bacterial cells, including those of species of the genera Escherichia, Bacillus,
- Serratia Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces) or eukaryotic (including fungi (especially yeast's), plants, protozoans and other parasites, and animals including insects (particularly Drosophila spp. cells), nematodes (particularly Caenorhabditis elegans cells), and mammals (particularly human cells)).
- the invention may be used in combination with methods for amplifying or sequencing nucleic acid molecules.
- Nucleic acid amplification methods may include the use of one or more polypeptides having reverse transcriptase activity, in methods generally known in the art as one-step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR) reverse transcriptase-amplification reactions.
- one-step e.g., one-step RT-PCR
- two-step e.g., two-step RT-PCR
- Amplification methods according to the invention may comprise one or more steps (e.g., two, three, four, five, seven, ten, etc.).
- the invention provides a method for amplifying a nucleic acid molecule comprising
- nucleic acid molecules complementary to all or a portion of the templates are amplified by such methods. If desired, recombination sites may be added to such amplified molecules during or after the amplification process (see, e.g., U.S. Patent Application No. 09/177,387 filed 10/23/98 based on U.S. provisional patent application no. 60/065,930 filed October 24, 1997).
- amplification and analysis of nucleic acid molecules or fragments are well known to one of ordinary skill in the art (see, e.g. , U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; Innis, M.A., et al, eds., PCR Protocols: A Guide to Methods and Applications, San Diego, California: Academic Press, Inc. (1990); Griffin, H.G., and Griffin, A.M., eds., PCR Technology: Current Innovations, Boca Raton, Florida: CRC Press (1994)).
- amplification methods which may be used in accordance with the present invention include PCR (U.S. Patent Nos.
- these amplification methods comprise: (a) mixing one or more enzymes with polymerase activity with the nucleic acid sample in the presence of one or more primers, and (b) amplifying the nucleic acid sample to generate a collection of amplified nucleic acid fragments, preferably by PCR or equivalent automated amplification technique.
- the amplified or synthesized nucleic acid fragments may be isolated for further use or characterization. This step is usually accomplished by separation of the amplified or synthesized nucleic acid fragments by size or by any physical or biochemical means including gel electrophoresis, capillary electrophoresis, chromatography (including sizing, affinity and immunochromatography), density gradient centrifugation and irnmunoadsorption. Separation of nucleic acid fragments by gel electrophoresis is particularly preferred, as it provides a rapid and highly reproducible means of sensitive separation of a multitude of nucleic acid fragments, and permits direct, simultaneous comparison of the fragments in several samples of nucleic acids.
- the invention is also directed to isolated nucleic acid molecules produced by the amplification or synthesis methods of the invention.
- one or more of the amplified or synthesized nucleic acid fragments are removed from the gel which was used for identification (see above), according to standard techniques such as electroelution or physical excision.
- the isolated unique nucleic acid fragments may then be inserted into standard vectors, including expression vectors, suitable for transfection or transformation of a variety of prokaryotic (bacterial) or eukaryotic (yeast, plant or animal including human and other mammalian) cells.
- nucleic acid molecules produced by the methods of the invention may be further characterized, for example by sequencing (i.e., determining the nucleotide sequence of the nucleic acid fragments), by methods described below and others that are standard in the art (see, e.g., U.S. Patent Nos. 4,962,022 and 5,498,523, which are directed to methods of DNA sequencing).
- Nucleic acid sequencing methods may comprise one or more steps.
- the invention may be combined with a method for sequencing a nucleic acid molecule comprising (a) mixing an enzyme with polymerase activity with a nucleic acid molecule to be sequenced, one or more primers, one or more nucleotides, and one or more terminating agents (such as a dideoxynucleotides) to form a mixture; (b) incubating the mixture under conditions sufficient to synthesize a population of molecules complementary to all or a portion of the molecule to be sequenced; and (c) separating the population to determine the nucleotide sequence of all or a portion of the molecule to be sequenced.
- a method for sequencing a nucleic acid molecule comprising (a) mixing an enzyme with polymerase activity with a nucleic acid molecule to be sequenced, one or more primers, one or more nucleotides, and one or more terminating agents (such as a dideoxynucleotides) to form
- Nucleic acid sequencing techniques which may be employed include dideoxy sequencing methods such as those disclosed in U.S. Patent Nos.
- kits which may be used in conjunction with the invention.
- Kits according to this aspect of the invention may comprise one or more containers, which may contain one or more components selected from the group consisting of one or more nucleic acid molecules or vectors of the invention, one or more primers, the molecules and/or compounds of the invention, supports of the invention, one or more polymerases, one or more reverse transcriptases, one or more recombination proteins (or other enzymes for carrying out the methods of the invention), one or more buffers, one or more detergents, one or more restriction endonucleases, one or more nucleotides, one or more terminating agents (e.g., ddNTPs), one or more transfection reagents, pyrophosphatase, and the like.
- nucleic acid molecules or vectors of the invention can be used with the invention. Further, due to the modularity of the invention, these nucleic acid molecules and vectors can be combined in wide range of ways.
- libraries can be supplied in kits of the invention.
- libraries may be in the form of replicable nucleic acid molecules or they may comprise nucleic acid molecules which are not associated with an origin of replication.
- nucleic acid molecules of libraries as well as other nucleic acid molecules, which are not associated with an origin of replication either could be inserted into other nucleic acid molecules which have an origin of replication or would be an expendable kit components.
- libraries supplied in kits of the invention may comprise two components: (1) the nucleic acid molecules of these libraries and (2) 5' and/or 3' recombination sites.
- nucleic acid molecules of a library when supplied with 5' and/or 3' recombination sites, it will be possible to insert these molecules into vectors, which also may be supplied as a kit component, using recombination reactions.
- recombination sites can be attached to the nucleic acid molecules of the libraries before use (e.g. , by the use of a ligase, which may also be supplied with the kit). In such cases, nucleic acid molecule which contain recombination sites or primers which can be used to generate recombination sites may be supplied with the kits.
- Vector supplied in kits of the invention can vary greatly. In most instances, these vectors will contain an origin of replication, at least one selectable marker, and at least one recombination site. For example, vectors supplied in kits of the invention can have four separate recombination sites which allow for insertion of nucleic acid molecules at two different locations. A vector of this type is shown schematically in Figure 6. Other attributes of vectors supplied in kits of the invention are described elsewhere herein.
- Kits of the invention can also be supplied with primers. These primers will generally be designed to anneal to molecules having specific nucleotide sequences. For example, these primers can be designed for use in PCR to amplify a particular nucleic acid molecule. Further, primers supplied with kits of the invention can be sequencing primers designed to hybridize to vector sequences. Thus, such primers will generally be supplied as part of a kit for sequencing nucleic acid molecules which have been inserted into a vector.
- One or more buffers may be supplied in kits of the invention. These buffers may be supplied at a working concentrations or may be supplied in concentrated form and then diluted to the working concentrations. These buffers will often contain salt, metal ions, co-factors, metal ion chelating agents, etc. for the enhancement of activities of the stabilization of either the buffer itself or molecules in the buffer. Further, these buffers may be supplied in dried or aqueous forms. When buffers are supplied in a dried form, they will generally be dissolved in water prior to use. Examples of buffers suitable for use in kits of the invention are set out in the following examples.
- Supports suitable for use with the invention may also be supplied with kits of the invention. Exemplary uses of supports in processes of the invention are shown in Figures 10-13.
- Kits of the invention may contain virtually any combination of the components set out above or described elsewhere herein. As one skilled in the art would recognize, the components supplied with kits of the invention will vary with the intended use for the kits. Thus, kits may be designed to perform various functions set out in this application and the components of such kits will vary accordingly.
- Two nucleic acid segments may be cloned in a single reaction using methods of the present invention.
- Methods of the present invention may comprise the steps of providing a first nucleic acid segment flanked by a first and a second recombination site, providing a second nucleic acid segment flanked by a third and a fourth recombination site, wherein either the first or the second recombination site is capable of recombining with either the third or the fourth recombination site, conducting a recombination reaction such that the two nucleic acid segments are recombined into a single nucleic acid molecule and cloning the single nucleic acid molecule.
- nucleic acid segments flanked by recombination sites may be provided.
- the nucleic acid segments may be provided either as discrete fragments or as part of a larger nucleic acid molecule and may be circular and optionally supercoiled or linear.
- the sites can be selected such that one member of a reactive pair of sites flanks each of the two segments.
- one nucleic acid molecule may comprise an ⁇ ttR site while the other comprises an ⁇ ttL site that reacts with the ⁇ ttR site.
- the products of an LR reaction are two molecules, one of which comprises an ⁇ ttB site and one of which comprises an ⁇ ttP site, it is possible to arrange the orientation of the starting ⁇ ttL and ⁇ ttR sites such that, after joining, the two starting nucleic acid segments are separated by a nucleic acid sequence that comprises either an ⁇ ttB site or an ⁇ ttP site.
- the sites may be arranged such that the two starting nucleic acid segments are separated by an ⁇ ttB site after the recombination reaction.
- recombination sites from other recombination systems may be used.
- one or more of the recombination sites may be a lox site or derivative.
- recombination sites from more than one recombination system may be used in the same construct.
- one or more of the recombination sites may be an att site while others may be lox sites.
- Various combinations of sites from different recombination systems may occur to those skilled in the art and such combinations are deemed to be within the scope of the present invention.
- nucleic acid segment A may be flanked by recombination sites having unique specificity, for example ⁇ ttLl and ⁇ ttL3 sites and nucleic acid segment B (DNA-B) may be flanked by recombination sites ⁇ ttR3 and ⁇ ttL2.
- the segments are indicated as DNA. This should not be construed as limiting the nucleic acids used in the practice of the present invention to DNA to the exclusion of other nucleic acids.
- the designation of the recombination sites is merely intend to convey that the recombination sites used have different specificities and should not be construed as limiting the invention to the use of the specifically recited sites.
- One skilled in the art could readily substitute other pairs of sites for those specifically exemplified.
- the ⁇ ttR3 and ⁇ ttL3 sites comprise a reactive pair of sites.
- Other pairs of unique recombination sites may be used to flank the nucleic acid segments.
- lox sites could be used as one reactive pair while another reactive pair may be att sites and suitable recombination proteins included in the reaction.
- the recombination sites discussed above can be used in various combinations.
- the only critical feature is that, of the recombination sites flanking each segment, one member of a reactive pair of sites, in this example an LR pair L3 and R3, is present on one nucleic acid segment and the other member of the reactive pair is present on the other nucleic acid segment.
- the two segments may be contacted with the appropriate enzymes and a
- the Destination Vector comprises a suitable selectable marker flanked by two recombination sites.
- the selectable marker may be a negative selectable marker (such as a toxic gene, e.g., ccdB).
- One site in the Destination Vector will be compatible with one site present on one of the nucleic acid segments while the other compatible site present in the Destination Vector will be present on the other nucleic acid segment.
- neither starting nucleic acid segment has recombination sites compatible with both the sites in the Destination Vector. Thus, neither starting nucleic acid segment can replace the selectable marker present in the Destination Vector.
- the reaction mixture may be incubated at about 25 °C for from about 60 minutes to about 16 hours. All or a portion of the reaction mixture will be used to transform competent microorganisms and the microorganisms screened for the presence of the desired construct.
- the Destination Vector comprises a negative selectable marker and the microorganisms transformed are susceptible to the negative selectable marker present on the Destination Vector.
- microorganisms will be grown under conditions permitting the negative selection against microorganisms not containing the desired recombination product.
- the resulting desired product consists of DNA-A and DNA-B separated by an ⁇ ttB3 site and cloned into the Destination Vector backbone.
- the same type of reaction /. e. , an LR reaction
- segment A With reference to Figure 2, if the orientation of segment A with respect to segment B were not critical, segment A could be flanked by LI sites on both ends oriented as inverted repeats and the end of segment B to be joined to segment A could be equipped with an RI site. This might be useful in generating additional complexity in the formation of combinatorial libraries between 0 segments A and B. That is, the joining of the segments can occur in various orientations and given that one or both segments joined may be derived from one or more libraries, a new population or library comprising hybrid molecules in random orientations may be constructed according to the invention.
- the recombination between the two 5 starting nucleic acid segments is shown as occurring before the recombination reactions with the Destination Vector, the order of the recombination reactions is not important. Thus, in some embodiments, it may be desirable to conduct the recombination reaction between the segments and isolate the combined segments.
- the combined segments can be used directly, for example, may be amplified, sequenced or used as linear expression elements as taught by Sykes, et al (Nature Biotechnology 17:355-359, 1999).
- the joined segments may be encapsulated as taught by Tawfik, et ⁇ /. (Nature Biotechnology 16:652-656, 1998) and subsequently assayed for one or more desirable properties.
- the combined segments may be used for in vitro expression of RNA by, for example, including a promoter such as the T7 promoter or SP6 promoter on one of the segments.
- a promoter such as the T7 promoter or SP6 promoter
- Such in vitro expressed RNA may optionally be translated in an in vitro translation system such as rabbit reticulocyte lysate.
- the joined segments may be further reacted with a Destination
- Vector resulting in the insertion of the combined segments into the vector may be desirable to isolate an intermediate comprising one of the segments and the vector.
- all three recombination reactions preferably occur (i.e., the reaction between segment A and the Destination Vector, the reaction between segment B and the Destination Vector, and the reaction between segment A and segment B) in order to produce a nucleic acid molecule in which both of the two starting nucleic acid segments are now joined in a single molecule.
- recombination sites may be selected such that, after insertion into the vector, the recombination sites flanking the joined segments form a reactive pair of sites and the joined segments may be excised from the vector by reaction of the flanking sites with suitable recombination proteins.
- the recombination reaction would produce an ⁇ ttP 1 site in the vector.
- the ⁇ ttPl site would then be capable of reaction with the ⁇ ttB 1 site on the other end of the joined segments.
- the joined segments could be excised using the recombination proteins appropriate for a BP reaction.
- each of the nucleic acid segments in Figure 2 may represent libraries, each of which may have a known or unknown nucleic acid sequence to be screened.
- one or more of the segments may have a sequence encoding one or more permutations of the amino acid sequence of a given peptide, polypeptide or protein.
- each segment may have a sequence that encodes a protein domain or a library representing various permutations of the sequence of protein domain. For example, one segment may represent a library of mutated forms of the variable domain of an antibody light chain while the other segment represents a library of mutated forms of an antibody heavy chain.
- DNA-A may comprise a regulatory sequence for directing expression (i.e., a promoter) and a sequence encoding a purification tag.
- Suitable purification tags include, but are not limited to, glutathione S-transferase (GST), the maltose binding protein (MBP), epitopes, defined amino acid sequences such as epitopes, haptens, six histidines (HIS6), and the like.
- GST glutathione S-transferase
- MBP maltose binding protein
- epitopes defined amino acid sequences such as epitopes, haptens, six histidines (HIS6), and the like.
- DNA-B may comprise a library of mutated forms of a protein of interest. The resultant constructs could be assayed for a desired characteristic such as enzymatic activity or ligand binding.
- DNA-B might comprise the common portion of the resulting fusion molecule.
- the above described methods may be used to facilitate the fusion of promoter regions or transcription termination signals to the 5 '-end or 3 '-end of structural genes, respectively, to create expression cassettes designed for expression in different cellular contexts, for example, by adding a tissue-specific promoter to a structural gene.
- one or more of the segments may represent a sequence encoding members of a random peptide library. This approach might be used, for example, to generate a population of molecules with a certain desirable characteristic.
- one segment might contain a sequence coding for a DNA binding domain while the other segment represents a random protein library. The resulting population might be screened for the ability to modulate the expression of a target gene of interest.
- both segments may represent sequences encoding members of a random protein library and the resultant synthetic proteins (e.g., fusion proteins) could be assayed for any desirable characteristic such as, for example, binding a specific ligand or receptor or possessing some enzymatic activity.
- nucleic acid segments encode an amino acid sequence.
- both of the segments may direct the transcription of an RNA molecule that is not translated into protein. This will be useful for the construction of tRNA molecules, ribozymes and anti-sense molecules.
- one segment may direct the transcription of an untranslated RNA molecule while the other codes for a protein.
- DNA-A may direct the transcription of an untranslated leader sequence that enhances protein expression such as the encephalomyocarditis virus leader sequence (EMC leader) while DNA-B encodes a peptide, polypeptide or protein of interest.
- a segment comprising a leader sequence might further comprise a sequence encoding an amino acid sequence.
- DNA-A might have a nucleic acid sequence corresponding to an EMC leader sequence and a purification tag while DNA-B has a nucleic acid sequence encoding a peptide, polypeptide or protein of interest.
- the above process is especially useful for the preparation of combinatorial libraries of single-chain antigen-binding proteins.
- Methods for preparing single-chain antigen-binding proteins are known in the art. (See, e.g., PCT Publication No.
- DNA-A could encode, for example, mutated forms of the variable domain of an antibody light chain and DNA-B could encode, for example, mutated forms of the variable domain of an antibody light chain.
- DNA-B could encode, for example, mutated forms of the variable domain of an antibody light chain.
- the intervening nucleic acid between DNA-A and DNA-B could encode a peptide linker for connecting the light and heavy chains. Cells which express the single-chain antigen-binding proteins can then be screened to identify those which produce molecules that bind to a particular antigen.
- single-chain antigen-binding proteins composed of two antibody light chains and two antibody heavy chains can also be produced. These single-chain antigen-binding proteins can be designed to associate and form multivalent antigen binding complexes. Using the constructs shown in
- DNA-A and DNA-B could each encode, for example, mutated forms of the variable domain of an antibody light chain.
- DNA-A and DNA-B could each encode, for example, mutated forms of the variable domain of an antibody heavy chain.
- Cells which express both single-chain antigen-binding proteins could then be screened to identify, for example, those which produce multivalent antigen-binding complexes having specificity for a particular antigen.
- the methods of the invention can be used, for example, to prepare and screen combinatorial libraries to identify cells which produce antigen-binding proteins (e.g., antibodies and/or antibody fragments or antibody fragment complexes comprising variable heavy or variable light domains) having specificities for particular epitopes.
- antigen-binding proteins e.g., antibodies and/or antibody fragments or antibody fragment complexes comprising variable heavy or variable light domains
- the methods of the invention also methods for preparing antigen-binding proteins and antigen-binding proteins prepared by the methods of the invention.
- Example 2 Simultaneous Cloning of Two Nucleic Acid Fragments Using an LR Reaction to Join the Segments and a BP Reaction to Insert the Segments into a Vector
- a first nucleic acid segment flanked by an ⁇ ttB recombination site and an ⁇ ttL recombination site may be joined to a second nucleic acid segment flanked by an ⁇ ttR recombination site that is compatible with the ⁇ ttL site present on the first nucleic acid segment and flanked by an ⁇ ttB site that may be the same or different as the ⁇ ttB site present on the first segment.
- Figure 3 shows an embodiment wherein the two ⁇ ttB sites are different. The two segments may be contacted with a vector containing ⁇ ttP sites in a BP reaction.
- a subsequent LR reaction would generate a product consisting of DNA- A and DNA-B separated by either an ⁇ ttP site or an ⁇ ttB site (the product of the LR reaction) and cloned into the vector backbone.
- the ⁇ ttL and ⁇ ttR sites are arranged so as to generate an ⁇ ttB site between the segments upon recombination.
- the ⁇ ttL and the ⁇ ttR may be oriented differently so as to produce an ⁇ ttP site between the segments upon recombination.
- the two segments may be separated by an ⁇ ttB site.
- each fragment may be present in a molar ratio of from about 25:1 to about 1 :25 vecto ⁇ fragment. In some embodiments, one or more of the fragments may be present at a molar ratio of from about 10:1 to l:10vector:fragment. Inapreferred embodiment, each fragment may be present at a molar ratio of about 1 : 1 vecto ⁇ fragment.
- the nucleic acid may be dissolved in an aqueous buffer and added to the reaction mixture.
- One suitable set of conditions is 4 ⁇ l CLONASETM enzyme mixture (e.g., Invitrogen Corp., Life Technologies Division, Cat. Nos. 11791-019 and 11789-013), 4 ⁇ l 5X reaction buffer and nucleic acid and water to a final volume of 20 ⁇ l. This will typically result in the inclusion of about 200 ng of Int and about 80 ng of IHF in a 20 ⁇ l BP reaction and about 150 ng Int, about 25 ng IHF and about 30 ng Xis in a 20 ⁇ l LR reaction.
- the final reaction mixture may include about 50mM Tris HCl, pH 7.5, about ImM EDTA, about lmg/ml BSA, about 75mM
- the final reaction mixture may include about 25mM Tris HCl, pH 7.5, about 5mM EDTA, about lmg/ml bovine serum albumin (BSA), about 22mM NaCl, and about 5 mM spermidine.
- the BP reaction can be conducted first and then the reaction conditions adjusted to about 50 mM NaCl, about 3.8 mM spermidine, about 3.4 mM EDTA and about 0.7 mg/ml by the addition of the LR CLONASETM enzymes and concentrated NaCl.
- the reaction solution may be incubated at suitable temperature such as, for example, 25 °C for from about 60 minutes to 16 hours. After the recombination reaction, the solution may be used to transform competent host cells and the host cells screened as described above.
- One example of a "one-tube" reaction protocol which facilitates the transfer of PCR products directly to Expression Clones in a two-step reaction performed in a single tube follows. This process can also be used to transfer a gene from one Expression Clone plasmid backbone to another.
- the Expression Clone is first be linearized within the plasmid backbone to achieve the optimal topology for the BP reaction and to eliminate false-positive colonies due to co-transformation.
- Twenty-five ⁇ l BP reaction mixture is prepared in a 1.5 ml tube with the following components:
- ⁇ ttB DNA 100-200 ng
- ⁇ ttP DNA 150 ng/ ⁇ l
- BP Reaction Buffer 5.0 ⁇ l
- the PCR product is amplified from a plasmid template containing selectable markers present on the GATEWAYTM pDONR or pDEST vectors (i.e., kan r or amp r ), the PCR product may be treated with the restriction endonuclease Dpnl to degrade the plasmid.
- selectable markers present on the GATEWAYTM pDONR or pDEST vectors i.e., kan r or amp r
- the PCR product may be treated with the restriction endonuclease Dpnl to degrade the plasmid.
- Such plasmids are a potential source of false- positive colonies in the transformation of GATEWAYTM reactions.
- the template for PCR or starting Expression Clone has the same selectable marker as the final Destination Vector (e.g., amp r )
- plating on LB plates containing 100 ⁇ g/ml ampicillin can be used to determine the amount of false positive colonies carried over to the LR reaction step.
- the mixture is then incubate at 25°C for 2 hours, after which 3 ⁇ l of proteinase K solution, followed by a further incubation of 10 minutes at 37°C. 1-2 ⁇ l of this mixtures is then used to transform 100 ⁇ l competent cells, which are then plated on LB plates containing 100 ⁇ g/ml ampicillin.
- Example 3 Cloning of PCR Products Using Fragments by Converting attB Sites into a Reactive Pair of att and attR Sites in a BP
- a similar strategy to that described in Example 2 can be used to recombine two PCR products and clone them simultaneously into a vector backbone. Since ⁇ ttL and ⁇ ttR sites are 100 and 125 base pairs long, respectively, it may be desirable to incorporate ⁇ ttB sites into the PCR primers since an ⁇ ttB site is 25 base pairs in length. Depending on the orientation of the ⁇ ttB site with respect to the nucleic acid segment being transferred, ⁇ ttB sites can be converted to either an ⁇ ttL or ⁇ ttR site by the BP reaction.
- the orientation of the ⁇ ttB site in the ⁇ ttB PCR primer determines whether the ⁇ ttB site is converted to ⁇ ttL or ⁇ ttR.
- two segments e.g., PCR products
- segment A flanked by mutated ⁇ ttB sites each having a different specificity e.g.
- segment B flanked by mutated ⁇ ttB sites of different specificity, wherein one of the ⁇ ttB sites present on segment A is the same as one of the ⁇ ttB sites present on segment B (e.g., segment B may contain ⁇ ttB3 and ⁇ ttB2 sites) may be joined and inserted into a vector.
- the segments may be reacted either individually or together with two ⁇ ttP site containing vectors in a BP reaction. Alternatively, the ⁇ ttP sites might be present on linear segments.
- One vector contains ⁇ ttP sites compatible with the ⁇ ttB sites present on segment A (e.g., ⁇ ttPl and ⁇ ttP3 sites).
- the other vector contains ⁇ ttP sites compatible with the ⁇ ttB sites present on segment B (e.g., attP3 and ⁇ ttP2 sites).
- each ⁇ ttP site may be provided on a segment.
- the orientations of the ⁇ ttB3 and ⁇ ttP3 sites are such that an ⁇ ttR3 site would be generated at the 5 '-end of the DNA-B segment and an ⁇ ttL3 site generated at the 3 '-end of segment A.
- the resulting entry clones are mixed with a Destination Vector in a subsequent LR reaction to generate a product consisting of DNA-A and DNA-B separated by an ⁇ ttB3 site and cloned into the Destination Vector backbone.
- This basic scheme has been used to link two segments, an ⁇ ttL 1 -fragment A- ⁇ ttL3 entry clone that is reacted with an ⁇ ttR3 -fragment B- ⁇ ttL2 entry clone, and to insert the linked fragments into the destination vector.
- two ⁇ ttP Donor vectors were constructed consisting of ⁇ ttPl-cc ⁇ - ⁇ ttP3 and ⁇ ttP3R-cc ⁇ T3- ⁇ ttP2 such that they could be reacted with appropriate ⁇ ttB PCR products in order to convert the ⁇ ttB sites to ⁇ ttL and ⁇ ttR sites.
- ⁇ ttP3R is used to indicated that the orientation of the ⁇ ttP3 site is such that reaction with a DNA segment having a cognate ⁇ ttB site will result in the production of an ⁇ ttR site on the segment.
- This is represented schematically in Figure 4 by the reversed orientation of the stippled and lined sections of the ⁇ ttB3 on segment B as compared to segment A. On segment B the stippled portion is adjacent to the segment while on segment A the lined portion is adjacent to the segment.
- This methodology was exemplified by constructing a DNA segment in which the tetracycline resistance gene (tet) was recombined with the ⁇ -galactosidase gene such that the two genes were separated by an ⁇ ttB site in the product.
- the tet gene was PCR amplified with 5 ' - ⁇ ttB 1 and 3 ' - ⁇ ttB 3 ends.
- the lacZ gene was PCR amplified with 5'- ⁇ ttB3R and 3'- ⁇ ttB2 ends.
- the two PCR products were precipitated with polyethylene glycol (PEG).
- the B 1 -tet-B3 PCR product was mixed with an ⁇ ttPl-cc ⁇ B- ⁇ ttP3 donor vector and reacted with BP CLONASETM using a standard protocol to generate an attL ⁇ -tet-attL3 entry clone.
- a correct tet entry clone was isolated and plasmid DNA prepared using standard techniques.
- the ⁇ ttB3R-/ ⁇ cZ- ⁇ ttB2 PCR product was mixed with an ⁇ ttP3R-cc ⁇ B- ⁇ ttP2 donor vector and reacted with BP CLONASETM to generate an ⁇ ttR3-/ ⁇ cZ- ⁇ ttL2 entry clone.
- an LR CLONASETM reaction was prepared in a reaction volume of 20 ⁇ l containing the following components: 60 ng (25 frnoles) of the supercoiled tet entry clone; 75 ng (20 fmoles) of the supercoiled lacZ entry clone; 150 ng (35 frnoles) of pDEST6 (described in PCT Publication WO 00/52027, the entire disclosure of which is incorporated herein by reference) linearized with Ncol; 4 ⁇ l reaction buffer and 4 ⁇ l of LR CLONASETM.
- the final reaction mixture contained 51 mM Tris-HCl,
- two segments can be reacted with a vector containing a single recombination site in order to convert one of the recombination sites on the segments into a different recombination site.
- segments containing ⁇ ttB sites may be reacted with a target vector having ⁇ ttP sites.
- segments A and B are reacted either together or separately with a vector having an ⁇ ttP3 site in order to convert the ⁇ ttB3 sites on the segments into an ⁇ ttL3 and an ⁇ ttR3, respectively. This is done so that the subsequent LR reaction between the two segments results in their being joined by an ⁇ ttB site.
- the segments may be joined with the ⁇ ttP site containing vector before, simultaneously with or after the recombination reaction to convert the sites to generate a co-integrate molecule consisting of DNA-A flanked by ⁇ ttLl and ⁇ ttL3 and DNA-B flanked by ⁇ ttR3 and ⁇ ttL2.
- a subsequent LR reaction will generate a product clone consisting of DNA-A and
- DNA-B separated by ⁇ ttB3 cloned into a vector backbone DNA-B separated by ⁇ ttB3 cloned into a vector backbone.
- an ⁇ ttP site designed to convert the ⁇ ttB used to link the segments to a reactive pair of ⁇ ttL and ⁇ ttR sites may be provided as shorter segments such as restriction fragments, duplexes of synthetic oligonucleotides or PCR fragments. Reactions involving a linear fragment in a
- BP reaction may require longer incubation times, such a overnight incubation.
- ⁇ ttB sites to ⁇ ttL or ⁇ ttR sites can also be accomplished solely by PCR.
- PCR primers containing ⁇ ttL or ⁇ ttR sites can be used to amplify a segment having an ⁇ ttB site on the end. Since the sequence of ⁇ ttL and ⁇ ttR sites contains a portion of the sequence of an ⁇ ttB site, the ⁇ ttB site in this case serves as an overlap region to which the ⁇ ttL or ⁇ ttR PCR primer can anneal.
- the primers for the PCR reaction may be provided as single stranded oligonucleotides.
- the primers may be provided as a duplex, for example, as the product of a PCR reaction to amplify either an ⁇ ttL or ⁇ ttR site.
- Two or more nucleic acid fragments can be cloned simultaneously into different regions of a vector having multiple sets of recombination sites each flanking a selectable marker.
- one or more of the selectable markers may be a negative selectable marker.
- nucleic acid segment A flanked by recombination sites that do not recombine with each other (e.g., ⁇ ttLl and ⁇ ttL2) and nucleic acid segment B
- (DNA-B) flanked by recombination sites that do not recombine with each other and do not recombine with the sites flanking segment A may be combined with a Destination Vector in an LR reaction.
- the Destination Vector will contain two pairs of recombination sites, each pair selected to recombine with the sites flanking one of the segments.
- Figure 6 shows two pairs of ⁇ ttR sites ( ⁇ ttRl / ⁇ ttR2 and ⁇ ttR3/ ⁇ ttR4) each flanking a ccdB negative selectable marker.
- the three nucleic acids can be combined in a single LR reaction.
- the resulting product will consist of DNA-A and DNA-B flanked by pairs of ⁇ ttB sites and cloned into distinct regions of the Destination Vector.
- an analogous method for inserting nucleic acid segments into a vector can be accomplished using a BP reaction.
- DNA-A flanked by recombination sites ⁇ ttB 1 and ⁇ ttB2 can be combined with
- the resulting product would consist of DNA-A and DNA-B cloned between pairs of ⁇ ttL sites into distinct regions of the vector.
- one segment may be flanked by lox sites while the other segment is flanked by att sites.
- a segment may have a lox site on one end and an att site on the other end or an frt site on one end.
- lox sites may be envisioned by those skilled in the art and such combinations are within the scope of the present invention.
- the intermediate might be used as is or might serve as the substrate in a subsequent recombination reaction to insert the second segment.
- the present invention is a method of cloning n nucleic acid segments, wherein n is an integer greater than 1 , comprising the steps of providing n nucleic acid segments, each segment flanked by two unique recombination sites, providing a vector comprising 2n recombination sites wherein each of the 2n recombination sites is capable of recombining with one of the recombination sites flanking one of the nucleic acid segments and conducting a recombination reaction such that the n nucleic acid segments are ' recombined into the vector thereby cloning the n nucleic acid segments.
- the vector comprises n copies of a selectable marker each copy flanked by two recombination sites. In other embodiments, the vector comprises two or more different selectable markers each flanked by two recombination sites. In some embodiments, one or more of the selectable markers may be a negative selectable marker.
- the present invention provides a method of cloning, comprising the steps of providing a first, a second and a third nucleic acid segment, wherein the first nucleic acid segment is flanked by a first and a second recombination site, the second nucleic acid segment is flanked by a third and a fourth recombination site and the third nucleic acid segment is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and none of the first, fourth, fifth or sixth recombination sites is capable of recombining with any of the first through sixth recombination sites, providing a vector comprising a seventh and an eighth recombination site flanking a first selectable marker and comprising a ninth and a tenth recombination site flanking a second selectable marker wherein none of the seventh through tenth recombination sites can recom
- a nucleic acid segment may comprise a sequence that functions as a promoter.
- the first and the second nucleic acid segments may comprise a sequence encoding a polypeptide and the recombination places both polypeptides in the same reading frame.
- a nucleic acid segment may comprise a sequence that functions as a transcription termination sequence.
- Both the inserted nucleic acid segments and the vector can contain sequences selected so as to confer desired characteristics on the product molecules.
- one or more of the portions of the vector adjacent to the inserted segments as well as the portion of the vector separating the inserted segments can contain one or more selected sequences.
- the selected sequences might encode ribozymes, epitope tags, structural domains, selectable markers, internal ribosome entry sequences, promoters, enhancers, recombination sites and the like.
- the portion of the vector separating the inserted segments may comprise one or more selectable markers flanked by a reactive pair of recombination sites in addition to the recombination sites used to insert the nucleic acid segments.
- the segment of the vector between the pairs of recombination sites may encode one or more a selectable markers such as the neomycin resistance gene.
- Segments A and B may contain nucleic acid sequences selected so as to be identical or substantially identical to a portion of a gene target that is to be disrupted.
- the Destination Vector will contain two portions of a gene of interest flanking a positive selectable marker.
- the vector can then be inserted into a cell using any conventional technology, such as transfection, whereupon the portions of the gene of interest present on the vector can recombine with the homologous portions of the genomic copy of the gene.
- Cells containing the inserted vector can be selected based upon one or more characteristics conferred by the selectable marker, for example, in the case when the selectable marker is the neomycin resistance gene, their resistance to G-418.
- one or more a negative selectable markers may be included in the portion of the Destination Vector that does not contain the target gene segments and the positive selectable marker. The presence of one or more negative selectable markers permits the selection against cells in which the entire
- Destination Vector was inserted into the genome or against cells in which the Destination Vector is maintained extrachromosomally.
- additional recombination sites may be positioned adjacent to the recombination sites used to insert the nucleic acid segments. Molecules of this type will be useful in gene targeting application where it is desirable to remove the selectable marker from the targeted gene after targeting, the so called “hit and run” methodology. Those skilled in the art will appreciate that the segments containing homologous sequence need not necessarily correspond to the sequence of a gene. In some instances, the sequences may be selected to be homologous to a chromosomal location other than a gene.
- expression vectors containing bi-cistronic expression elements where two structural genes are expressed from a single promoter and are separated by an internal ribosome entry sequence
- nucleic acid segments and recombination sites of the same specificity can be used on both ends of the segment.
- segment A could be flanked by LI sites on both ends and the vector equipped with two RI sites. This might be useful in generating additional complexity in the formation of combinatorial libraries between segments A and B.
- the present invention provides a method of cloning n nucleic acid segments, wherein n is an integer greater than 1, comprising the steps of providing a 1 st through an n* nucleic acid segment, each segment flanked by two unique recombination sites, wherein the recombination sites are selected such that one of the two recombination sites flanking the I th segment, n i5 reacts with one of the recombination sites flanking the segment and the other recombination site flanking the I th segment reacts with one of the recombination sites flanking the n i+1 th segment, providing a vector comprising at least two recombination sites wherein one of the two recombination sites on the vector reacts with one of the sites on the 1 st nucleic acid segment and another site on the vector reacts with a recombination site on the n* nucleic acid segment.
- At least two nucleic acid segments comprise sequences encoding a polypeptide and the recombination places both polypeptides in the same reading frame.
- at least one nucleic acid segment comprises a sequence that functions as a transcription termination sequence.
- at least one fragment comprises an origin of replication.
- at least one fragment comprises a sequence coding for a selectable marker.
- the present invention provides a method of cloning, comprising the steps of providing a first, a second and a third nucleic acid segment, wherein the first nucleic acid segment is flanked by a first and a second recombination site, the second nucleic acid segment is flanked by a third and a fourth recombination site and the third nucleic acid segment is flanked by a fifth and a sixth recombination site, wherein the second recombination site is capable of recombining with the third recombination site and the fourth recombination site is capable of recombining with the fifth recombination site, providing a vector comprising a seventh and an eighth recombination site and conducting at least one recombination reaction such that the second and the third recombination sites recombine and the fourth and the fifth recombination sites recombine and
- the invention may be modified by placing recombination sites having the same specificity on both ends of the given segment and adjusting the recombination sites of the adj acent segments and/or the recombination sites in the vector accordingly.
- the invention provides a modular method for the construction of vectors.
- At least one nucleic acid segment comprises a sequence that functions as a promoter. In some embodiments, at least two nucleic acid segments comprise a sequence encoding a polypeptide and the recombination places both polypeptides in the same reading frame. In some embodiments, at least one nucleic acid segment comprises a sequence that functions as a transcription termination sequence. In some embodiments, at least one fragment comprises an origin of replication. In some embodiments, at least one fragment comprises a sequence coding for a selectable marker. In some embodiments, a fragment may comprise sequence coding for more than one function. In some embodiments, a fragment may comprise sequence coding for an origin of replication and sequence encoding a selectable marker.
- Figure 20A shows one example of a vector which contains two inserted DNA segments, the expression of which is driven by different promoters (i.e., two different T7 promoters).
- RNA interference double-stranded RNA
- Methods of the invention can be used in a number of ways to produce molecules such as RNAi.
- expression products of nucleic acid molecules of the invention can be used to silence gene expression.
- Figure 20B One example of a construct designed to produce RNAi is shown in Figure 20B. In this construct, a DNA segment is inserted into a vector such that RNA corresponding to both strands are produced as two separate transcripts.
- Figure 20C Another example of a construct designed to produce RNAi. In this construct, two copies of a DNA segment are inserted into a vector such that
- RNA corresponding to both strands are again produced.
- Figure 20D Yet another example of a construct designed to produce RNAi is shown in Figure 20D.
- two copies of a DNA segment are inserted into a vector such that RNA corresponding to both strands are produced as a single transcript.
- the exemplary vector system shown in shown in Figure 20E comprises two vectors, each of which contain copies of the same DNA segment. Expression of one of these DNA segments results in the production of sense RNA while expression of the other results in the production of an anti-sense RNA.
- RNA strands produced from vectors represented in Figures 20B-20E will thus have complementary nucleotide sequences and will generally hybridize either to each or intramolecularly under physiological conditions.
- Nucleic acid segments designed to produce RNAi need not correspond to the full-length gene or open reading frame.
- the segment may only correspond to part of the ORF (e.g., 50 nucleotides at the 5' or 3' end of the ORF).
- Figures 20B-20E show vectors designed to produce RNAi
- nucleic acid segments may also perform the same function in other forms (e.g. , when inserted into the chromosome of a host cell).
- Gene silencing methods involving the use of compounds such as RNAi and antisense RNA, for examples, are particularly useful for identifying gene functions.
- gene silencing methods can be used to reduce or prevent the expression of one or more genes in a cell or organism. Phenotypic manifestations associated with the selective inhibition of gene functions can then be used to assign role to the "silenced" gene or genes. As an example, Chuang etal, Proc. Natl. Acad. Sci. (USA) 97:4985-4990 (2000), have demonstrated that in vivo production of RNAi can alter gene activity in Arabidopsis thaliana. Thus, the invention provides methods for regulating expression of nucleic acid molecules in cells and tissues comprising the expression of RNAi and antisense
- the invention further provides methods for preparing nucleic acid molecules which can be used to produce RNA corresponding to one or both strands of a DNA molecule.
- the invention relates to compounds and methods for gene silencing involving ribozymes.
- the invention provides antisense
- RNA/ribozymes fiisions which comprise (1) antisense RNA corresponding to a target gene and (2) one or more ribozymes which cleave RNA (e.g. , hammerhead ribozyme, hairpin ribozyme, delta ribozyme, Tetrahymena L-21 ribozyme, etc.).
- ribozymes which cleave RNA (e.g. , hammerhead ribozyme, hairpin ribozyme, delta ribozyme, Tetrahymena L-21 ribozyme, etc.).
- vectors which express these fusions, methods for producing these vectors, and methods for using these vector to suppress gene expression.
- a Destination Vector is constructed which encodes a ribozyme located next to a ccdB gene, wherein the a cc ⁇ B gene is flanked by ⁇ ttR sites.
- An LR reaction is used to replace the ccdB gene with a nucleic acid molecule which upon expression produces an antisense RNA molecule.
- the expression product will result in the production of an antisense sequence fused to the ribozyme by an intervening sequence encoded by an ⁇ ttB site.
- this ⁇ ttB site can be removed from the transcript (e.g., using intron and exon slice sequences), if desired, or, in certain cases, nucleic acid which encodes the ribozyme can be embedded in the ⁇ ttB site.
- antisense molecules fused to ribozymes can be used, for example, to cleave specific RNA molecules in a cell. This is so because the antisense RNA portion of the transcript can be designed to hybridize to particular mRNA molecules. Further, the ribozyme portion of the transcript can be designed to cleave the RNA molecule to which it has hybridized. For example, the ribozyme can be one which cleaves double-stranded RNA (e.g. , Tetrahymena L-21 ribozyme).
- the recently developed recombinational cloning techniques described above permit the rapid movement of a target nucleic acid from one vector background to one or more other vector backgrounds. Because the recombination event is site specific, the orientation and reading frame of the target nucleic acid can be controlled with respect to the vector. This control makes the construction of fusions between sequences present on the target nucleic acid and sequences present on the vector a simple matter.
- a gene may be expressed in four forms: native at both amino and carboxy termini, modified at either end, or modified at both ends.
- a construct containing the target gene of interest may include the N-terminal methionine ATG codon, and a stop codon at the carboxy end, of the open reading frame, or ORF, thus ATG - ORF - stop.
- the gene construct will include translation initiation sequences, tis, that may be located upstream of the ATG that allow expression of the gene, thus tis - ATG - ORF - stop. Constructs of this sort allow expression of a gene as a protein that contains the same amino and carboxy amino acids as in the native, uncloned, protein.
- RNA (lower case, italics): t/s7 - atg - tag - tis2 - atg - orf- stop
- the present invention meets this need by providing materials and methods for the controlled expression of a C- and/or N-terminal fusion to a target gene using one or more suppressor tRNAs to suppress the termination of translation at a stop codon.
- the present invention provides materials and methods in which a gene construct is prepared flanked with recombination sites.
- the construct is prepared with a sequence coding for a stop codon preferably at the C-terminus of the gene encoding the protein of interest.
- a stop codon can be located adjacent to the gene, for example, within the recombination site flanking the gene.
- the target gene construct can be transferred through recombination to various vectors which can provide various C-terminal or N-terminal tags (e.g. , GFP, GST, His Tag, GUS, etc.) to the gene of interest.
- C-terminal or N-terminal tags e.g. , GFP, GST, His Tag, GUS, etc.
- the suppressor tRNA when the suppressor tRNA is not expressed) while expression of the gene as a carboxy fusion protein occurs under suppressing conditions.
- the present invention is exemplified using an amber suppressor supF, which is a particular tyrosine tRNA gene (tyrT) mutated to recognize the UAG stop codon.
- tyrT tyrosine tRNA gene
- the gene coding for the suppressing tRNA has been incorporated into the vector from which the target gene is to be expressed.
- the gene for the suppressor tRNA may be in the genome of the host cell. In still other embodiments, the gene for the suppressor may be located on a separate vector and provided in trans. In embodiments of this type, the vector containing the suppressor gene may have an origin of replication selected so as to be compatible with the vector containing the gene construct.
- an appropriate vector for providing the suppressor tRNA in trans may include the selection of an appropriate antibiotic resistance marker.
- an appropriate antibiotic resistance marker for example, if the vector expressing the target gene contains an antibiotic resistance marker for one antibiotic, a vector used to provide a suppressor tRNA may encode resistance to a second antibiotic. This permits the selection for host cells containing both vectors.
- more than one copy of a suppressor tRNA may be provided in all of the embodiments described above.
- a host cell may be provided that contains multiple copies of a gene encoding the suppressor tRNA.
- multiple gene copies of the suppressor tRNA under the same or different promoters may be provided in the same vector background as the target gene of interest.
- multiple copies of a suppressor tRNA may be provided in a different vector than the one use to contain the target gene of interest.
- one or more copies of the suppressor tRNA gene may be provided on the vector containing the gene for the protein of interest and/or on another vector and/or in the genome of the host cell or in combinations of the above.
- the genes may be expressed from the same or different promoters which may be the same or different as the promoter used to express the gene encoding the protein of interest.
- two or more different suppressor tRNA genes may be provided.
- one or more of the individual suppressors may be provided in multiple copies and the number of copies of a particular suppressor tRNA gene may be the same or different as the number of copies of another suppressor tRNA gene.
- Each suppressor tRNA gene, independently of any other suppressor tRNA gene, may be provided on the vector used to express the gene of interest and/or on a different vector and/or in the genome of the host cell.
- a given tRNA gene may be provided in more than one place in some embodiments.
- a copy of the suppressor tRNA may be provided on the vector containing the gene of interest while one or more additional copies may be provided on an additional vector and/or in the genome of the host cell.
- the genes may be expressed from the same or different promoters which may be the same or different as the promoter used to express the gene encoding the protein of interest and may be the same or different as a promoter used to express a different tRNA gene.
- the GUS gene was cloned in frame with a
- the plasmid also contained a supF gene expressing a suppressor tRNA.
- the plasmid was introduced into a host cell where approximately 60 percent of the GUS gene was expressed as a fusion protein containing the GST tag.
- a plasmid containing the same GUS-stop codon-GST construct did not express a detectable amount of a fusion protein when expressed from a vector lacking the supF gene.
- the supF gene was expressed as part of the mRNA containing the GUS-GST fusion. Since tRNAs are generally processed from larger RNA molecules, constructs of this sort can be used to express the suppressor tRNAs of the present invention. In other embodiments, the RNA containing the tRNA sequence may be expressed separately from the mRNA containing the gene of interest.
- the target gene of interest and the gene expressing the suppressor tRNA may be controlled by the same promoter.
- the target gene of interest may be expressed from a different promoter than the suppressor tRNA.
- a regulatable promoter for example, either the target gene of interest and/or the gene expressing the suppressor tRNA may be controlled by a promoter such as the lac promoter or derivatives thereof such as the tac promoter.
- both the target gene of interest and the suppressor tRNA gene are expressed from the T7 RNA polymerase promoter. Induction of the T7 RNA polymerase turns on expression of both the gene of interest (GUS in this case) and the supF gene expressing the suppressor tRNA as part of one RNA molecule.
- the expression of the suppressor tRNA gene may be under the control of a different promoter from that of the gene of interest. In some embodiments, it may be possible to express the suppressor gene before the expression of the target gene. This would allow levels of suppressor to build up to a high level, before they are needed to allow expression of a fusion protein by suppression of a the stop codon.
- the target gene is controlled by the T7 RNA polymerase promoter and the expression of the T7 RNA polymerase is controlled by a promoter inducible with an inducing signal other than IPTG, e.g., NaCl
- a promoter inducible with an inducing signal other than IPTG e.g., NaCl
- the expression of the suppressor tRNA might be induced about 15 minutes to about one hour before the induction of the T7 RNA polymerase gene.
- the expression of the suppressor tRNA may be induced from about 15 minutes to about 30 minutes before induction ofthe T7 RNA polymerase gene.
- the expression of the T7 RNA polymerase gene is under the control of a salt inducible promoter.
- a cell line having an inducible copy of the T7 RNA polymerase gene under the control of a salt inducible promoter is commercially available from Invitrogen Corp., Life Technologies Division under the designation of the BL21 SI strain.
- the expression of the target gene of interest and the suppressor tRNA can be arranged in the form of a feedback loop.
- the target gene of interest may be placed under the control of the T7 RNA polymerase promoter while the suppressor gene is under the control of both the T7 promoter and the lac promoter, and the T7 RNA polymerase gene itself is transcribed by both the T7 promoter and the lac promoter, and the T7 RNA polymerase gene has an amber stop mutation replacing a normal tyrosine stop codon, e.g., the 28 th codon (out of 883). No active T7 RNA polymerase can be made before levels of suppressor are high enough to give significant suppression.
- the T7 polymerase expresses the suppressor gene as well as itself.
- only the suppressor gene is expressed from the T7 RNA polymerase promoter. Embodiments of this type would give a high level of suppressor without producing an excess amount of T7 RNA polymerase.
- the T7 RNA polymerase gene has more than one amber stop mutation (see, e.g., Figure 14B). This will require higher levels of suppressor before active T7 RNA polymerase is produced.
- a vector may be constructed so as to permit the regulatable expression of N- and/or C-terminal fusions of a protein of interest from the same construct.
- a first tag sequence, TAG1 in Figure 15 is expressed from a promoter represented by an arrow in the figure.
- the tag sequence includes a stop codon in the same reading frame as the tag.
- the stop codon 1 may be located anywhere in the tag sequence and is preferably located at or near the C-terminal of the tag sequence.
- the stop codon may also be located in the recombination site RS, or in the internal ribosome entry sequence (IRES).
- the construct also includes a gene of interest (GENE) which includes a stop codon 2.
- GENE gene of interest
- the first tag and the gene of interest are preferably in the same reading frame although inclusion of a sequence that causes frame shifting to bring the first tag into the same reading frame as the gene of interest is within the scope of the present invention.
- Stop codon 2 is in the same reading frame as the gene of interest and is preferably located at or near the end of the coding sequence for the gene. Stop codon 2 may optionally be located within the recombination site RS 2 .
- the construct also includes a second tag sequence in the same reading frame as the gene of interest indicated by TAG2 in Figure 15 and the second tag sequence may optionally include a stop codon 3 in the same reading frame as the second tag.
- a transcription terminator may be included in the construct after the coding sequence of the second tag (not shown in Figure 15). Stop codons 1, 2 and 3 may be the same or different. In some embodiments, stop codons 1, 2 and 3 are different. In embodiments where 1 and 2 are different, the same construct may be used to express an N-terminal fusion, a C-terminal fusion and the native protein by varying the expression of the appropriate suppressor tRNA. For example, to express the native protein, no suppressor tRNAs are expressed and protein translation is controlled by the IRES.
- a suppressor tRNA that suppresses stop codon 1 is expressed while a suppressor tRNA that suppresses stop codon 2 is expressed in order to produce a C-terminal fusion.
- a doubly tagged protein of interest in which case suppressor tRNAs that suppress both stop codon 1 and stop codon 2 may be expressed.
- Plasmid templates pEZC1301 and pEZC1313 (described in PCT Publication WO 00/52027, the entire disclosure of which is incorporated herein by reference), each containing a single wild-type att site, were used for the generation of PCR products containing ⁇ ttL or ⁇ ttR sites, respectively. Plasmid templates were linearized with 4/wNI, phenol extracted, ethanol precipitated and dissolved in TE to a concentration of 1 ng/ ⁇ l. PCR primers (capital letters represent base changes from wild-type V. ⁇ ttLl gggg agcct gctttttGtacAaaa gttggcatta taaaaagca ttgc (SEQ ID
- PCR primers were dissolved in TE to a concentration of 500 pmol/ ⁇ l.
- Primer mixes were prepared, consisting of ⁇ ttLl + ⁇ ttLright primers, ⁇ ttL2 + ⁇ ttLright primers, ⁇ ttRl + ⁇ ttRright primers, and ⁇ ttR2 + ⁇ ttRright primers, each mix containing 20 pmol/ ⁇ l of each primer.
- the resulting ⁇ ttL PCR product was 1.5 kb, and the resulting ⁇ ttR PCR product was 1.0 kb.
- PCR reactions were PEG/MgCl 2 precipitated by adding 150 ⁇ l H 2 O and 100 ⁇ l of 3x PEG/ MgCl 2 solution followed by centrifugation. The PCR products were dissolved in 50 ⁇ l of TE. Quantification of the PCR product was performed by gel electrophoresis of 1 ⁇ l and was estimated to be 50-100 ng/ ⁇ l.
- Clonase reactions were incubated at 25 °C for 2 hours. Two ⁇ l of proteinase K (2 mg/ml) was added to stop the reaction. Ten ⁇ l was then run on a 1 % agarose gel. Positive control reactions were performed by reacting ⁇ ttLl
- PCR product (1.0 kb) with ⁇ ttRl PCR product (1.5 kb) and by similarly reacting ⁇ ttL2 PCR product with ⁇ ttR2 PCR product to observe the formation of a larger (2.5 kb) recombination product.
- Negative controls were similarly performed by reacting ⁇ ttLl PCR product with ⁇ ttR2 PCR product and vice versa or reactions of ⁇ ttL PCR product with an ⁇ ttL plasmid, etc.
- Plasmids containing single att sites could also be used as recombination substrates in general to test all Entry and Destination vectors (/. e. , those containing ⁇ ttL, ⁇ ttR , ⁇ ttB and ⁇ ttP sites). This would eliminate the need to do PCR reactions.
- Destination and Entry plasmids when reacted with appropriate ⁇ tt-containing PCR products formed linear recombinant molecules that could be easily visualized on an agarose gel when compared to control reactions containing no ⁇ ttL or ⁇ ttR PCR product.
- the functionality of Destination and Entry vectors constructed according to the invention may be determined, for example, by carrying out the linearization assay described above.
- the cloning of PCR products using the GATEWAYTM PCR Cloning System requires the addition of ⁇ ttB sites ( ⁇ ttBl and ⁇ ttB 2) to the ends of gene-specific primers used in the PCR reaction.
- Available data suggested that the user add 29 bp (25 bp containing the ⁇ ttB site plus four G residues) to the gene-specific primer. It would be advantageous to high volume users of the GATEWAYTM PCR Cloning System to generate ⁇ ttB-containing PCR product using universal ⁇ ttB adapter-primers in combination with shorter gene-specific primers containing a specified overlap to the adapters.
- Bl-Hgb GGGG ACA AGT TTG TAC AAA AAA GCA GGC T-5'-Hgb* (SEQ ID NO:47)
- B2-Hgb GGGG ACC ACT TTG TAC AAG AAA GCT GGG T-3 ' -Hgb** (SEQ ID NO:48)
- HBl-Hgb A AAA GCA GGC T-5'-Hgb (SEQ ID NO: 55)
- 11B2-Hgb G AAA GCT GGG T-3 ' -Hgb
- AttBl adapter GGGG ACA AGT TTG TAC AAA AAA GCA GGC T
- the aim of these experiments was to develop a simple and efficient universal adapter PCR method to generate ⁇ ttB containing PCR products suitable for use in the GATEWAYTM PCR Cloning System.
- the reaction mixtures and thermocycling conditions should be simple and efficient so that the universal adapter PCR method could be routinely applicable to any PCR product cloning application.
- PCR reaction conditions were initially found that could successfully amplify predominately full-length PCR product using gene-specific primers containing 18 bp and 15 bp overlap with universal ⁇ ttB primers. These conditions are outlined below: 10 pmoles of gene-specific primers
- PCR products from reactions using the 18 bp, 15 bp, 12 bp, 11 bp and 10 bp overlap gene-specific primers were purified using the CONCERT® Rapid PCR Purification System (PCR products greater than 500 bp can be PEG precipitated).
- the purified PCR products were subsequently cloned into an ⁇ ttP containing plasmid vector using the GATEWAYTM PCR Cloning System (Invitrogen Corp., Life Technologies Division; Rockville, MD) and transformed into E. coli. Colonies were selected and counted on the appropriate antibiotic media and screened by PCR for correct inserts and orientation.
- Raw PCR products (unpurified) from the ⁇ ttB adapter PCR of a plasmid clone of part of the human beta-globin (Hgb) gene were also used in GATEWAYTM PCR Cloning System reactions.
- PCR products generated with the full ⁇ ttB Bl/B2-Hgb, the 12B1/B2, 11B1/B2 and 10B1/B2 ⁇ ttB overlap Hgb primers were successfully cloned into the GATEWAYTM pENTR21 ⁇ ttP vector (described in PCT Publication WO 00/52027, the entire disclosure of which is incorporated herein by reference).
- the cloning efficiency expressed as cfu/ml is shown below:
- PCR primers with 12 bp ⁇ ttB overlaps were used to amplify cDNAs of different sizes (ranging from 1 to 4 kb) from a leukocyte cDNA library and from first strand cDNA prepared from HeLa total RNA.
- ⁇ ttB PCR products can be generated with primers containing as few as 12 bases of ⁇ ttB added to template-specific primers using a two-step PCR protocol.
- template-specific primers containing 12 bases of ⁇ ttB are used in 10 cycles of PCR to amplify the target gene.
- a portion of this PCR reaction is transferred to a second PCR reaction containing universal ⁇ ttB adapter primers to amplify the full- ⁇ ttB PCR product.
- Template-specific primers with 12 bases of ⁇ ttBl and ⁇ ttB2 at their 5'- ends are designed as shown below:
- the template-specific part of the primers is generally be designed to have a Tm of greater than 50°C.
- the optimal annealing temperature is determined by the Tm of the template-specific part of the primer.
- ⁇ ttBl adapter primer GGGGACAAGTTTGTACAAAAAAGCAGGCT (SEQ ID NO:47)
- ⁇ ttB2 adapter primer GGGGNCCACTTTGTACAAGAAAGCTGGGT (SEQ ID ⁇ O:48)
- a 50 ⁇ l PCR reaction containing 10 pmoles of each template-specific primer and the appropriate amount of template DNA is prepared. Tubes containing this PCR reaction mixture are placed in a thermal cycler at 95 °C and incubated for 2 minutes. Ten cycles of PCR are performed as follows: Denature 94 °C for 15 seconds
- Ten ⁇ l of the PCR reaction product is transferred to a 40 ⁇ l PCR reaction mixture containing 40 pmoles each of the ⁇ ttBl and ⁇ ttB2 adapter primers. Tubes containing this mixtures are then placed in a thermal cycler at 95°C and incubated for 1 minute.
- the amplification products are then analyzed by agarose gel electrophoresis.
- the bacteriophage lambda ⁇ ttL and ⁇ ttR sites were systematically mutagenized and examined to define precisely which mutations produce unique changes in att site specificity.
- the determinants of specificity have previously been localized to the 7 bp overlap region (TTT AT AC, which is defined by the cut sites for the integrase protein and is the region where strand exchange takes place) within the 15 bp core region (GCTTTTTTATACTAA (SEQ ID NO:37)) that is identical in all four lambda att sites, ⁇ ttB, ⁇ ttP, ⁇ ttL and ⁇ ttR.
- mutant ⁇ ttL and ⁇ ttR sites were generated by PCR and tested in an in vitro site-specific recombination assay. In this way all possible single base pair changes within the 7 bp overlap region of the core att site were generated as well as five additional changes outside the 7 bp overlap but within the 15 bp core att site.
- PCR substrate was tested in the in vitro recombination assay with each of the ⁇ ttR PCR substrates.
- GATEWAYTM sites (note: ⁇ ttL2 sequence in GATEWAYTM plasmids begins “accca” while the ⁇ ttL2 site in this example begins “agccf'to reflect wild-type ⁇ ttL outside the core region.) :
- AttL2 gggg agcct gctttCttGtacAaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 42)
- Att TIC gggg agcct gctttCttatactaa gttggcatta taaaaa- agca ttgc (SEQ ID NO: 63)
- AttLTIG gggg agcct gctttGttatactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 64)
- AttLT2A gggg agcct gcttttAtatactaa gttggcatta taaaaa- agca ttgc (SEQ ID NO : 65 )
- AttLT2C gggg agcct gcttttCtatactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 66)
- AttLT2G gggg agcct gcttttGtatactaa gttggcatta taaaa- aagca ttgc (SEQ ID NO: 67)
- AttLT3A gggg agcct gcttttAatactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 68)
- AttLT3C gggg agcct gcttttCatactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 69)
- Att A4C gggg agcct gctttttCtactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 71)
- Att A4G gggg agcct gctttttCtactaa gttggcatta taaaa- aagca ttgc (SEQ ID NO: 72)
- Att A4T gggg agcct gctttttTtactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 73)
- AttIA6C gggg agcct gctttttatCctaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 77)
- AttLA6G gggg agcct gctttttatGctaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 78)
- AttLA6T gggg agcct gctttttatTctaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 79)
- AttLC7A gggg agcct gcttttttataAtaa gttggcatta taaaa- aagca ttgc (SEQ ID NO: 80)
- AttLC7G gggg agcct gcttttttataGtaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 81)
- AttLC7T gggg agcct gcttttttataTtaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 82)
- AttL9 gggg agcct gcCtttttatactaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 84)
- AttLlO gggg agcct gcttCtttatactaa gttggcatta taaaaagca ttgc (SEQ ID NO: 85)
- attL14 gggg agcct gcttttttatacCaa gttggcatta taaaaaagca ttgc (SEQ ID NO: 86)
- AttL15 gggg agcct gctttttatactaG gttggcatta taaaaaagca ttgc (SEQ ID NO: 87)
- CLONASETM reactions were incubated at 25°C for 2 hours.
- 2 ⁇ l of 1 OX CLONASETM stop solution proteinase K, 2 mg/ml
- 10 ⁇ l of the reaction mixtures were run on a 1 % agarose gel.
- TTTATAC changes in the last four positions
- BP reactions were carried out between 300 ng (100 frnoles) of pDONR201 (Invitrogen Corp., Life Technologies Division, Cat. No. 11798-014) with 80 ng (80 frnoles) of attB-tet PCR product in a 20 ⁇ l volume with incubation for 1.5 hours at 25 °C, creating pENTR201-tet Entry clones.
- Table 7 A comparison of the cloning efficiencies of the above-noted ⁇ ttB sites in BP reactions is shown in Table 7.
- the entry clones produced above were then transferred to pDEST20 (Invitrogen Corp., Life Technologies Division, Cat. No. 11807-013) via LR reactions (300 ng (64 frnoles) pDEST20 mixed with 50 ng (77 frnoles) of the respective pENTR201-tet Entry clone in 20 ⁇ l volume; incubated for a 1 hour incubation at 25 °C). The efficiencies of cloning for these reactions are compared in Table 8.
- AttBl GGGG ACAAGTTTGTACAAA AAAGC AGGCT (SEQ ID NO: 47) attBlnl ⁇ -20:
- the starting population size of degenerate att sites is 4 5 or 1024 molecules.
- Four different populations were transferred through two BP reactions and two LR reactions. Following transformation of each reaction, the population of transformants was amplified by growth in liquid media containing the appropriate selection antibiotic. DNA was prepared from the population of clones by alkaline lysis miniprep and used in the next reaction. The results of the BP and LR cloning reactions are shown below.
- Recombination sites used with the invention may also have embedded functions or properties.
- An embedded functionality is a function or property conferred by a nucleotide sequence in a recombination site which is not directly associated with recombination efficiency or specificity.
- recombination sites may contain protein coding sequences (e.g., intein coding sequences), intron/exon splice sites, origins of replication, and/or stop codons.
- protein coding sequences e.g., intein coding sequences
- intron/exon splice sites e.g., origins of replication, and/or stop codons.
- recombination sites will be more likely to have features (e.g., stop codons) which interfere with desired functions or properties. Further, recombination sites which have more than one (e.g., two, three, four, five, etc.) embedded functions or properties may also be prepared.
- the invention provides methods for removing nucleotide sequences encoded by recombination sites from RNA molecules.
- One example of such a method employs the use of intron/exon splice sites to remove RNA encoded by recombination sites from RNA transcripts.
- nucleotide sequences which encode these intron exon splice sites may be fully or partially embedded in the recombination sites which encode sequences excised from RNA molecules or these intron exon splice sites may be encoded by adjacent nucleic acid sequence.
- one intron exon splice sites may be encoded by recombination site and another intron exon splice sites may be encoded by other nucleotide sequences (e.g. , nucleic acid sequences of the vector or a nucleic acid of interest). Nucleic acid splicing is discussed in the following publications: R. Reed, Curr. Opin. Genet. Devel. 5:215-220(1996);
- RNA corresponding to recombination sites from RNA transcripts or amino acid residues encoded by recombination sites. Removal of such sequences can be performed in several ways and can occur at either the RNA or protein level.
- One instance where it will generally be advantageous to remove RNA transcribed from a recombination site will be where a nucleic acid molecule which an ORF is inserted into a vector in an orientation which is intended to result in the expression of a fusion protein (e.g., GFP) between amino acid residues encoded by the ORF and amino acid residues encoded by the vector (e.g., GFP).
- a fusion protein e.g., GFP
- the presence of an intervening recombination site between the ORF and the vector coding sequences may result in the recombination site (1) contributing codons to the mRNA which results in the inclusion of additional amino acid residues in the expression product, (2) contributing a stop codon to the mRNA which prevents the production of the desired fusion protein, and/or (3) shifting the reading frame of the mRNA such that the two protein are not fused "in-frame.”
- One method for removing recombination sites from mRNA molecules involves the use intron/exon splice sites (i.e., splice donor and splice acceptor sites). Splice sites can be suitably positioned in a number of locations.
- the first splice site could be encoded for by vector sequences located 3' to the GFP coding sequences and the second splice site could be partially embedded in the recombination site which separates the GFP coding sequences from the coding sequences of the ORF. Further, the second splice site either could abut the 3' end of the recombination site or could be positioned a short distance (e.g., 2, 4, 8, 10, 20 nucleotides) 3' to the recombination site. In addition, depending on the length of the recombination site, the second splice site could be fully embedded in the recombination site.
Abstract
Description
Claims
Priority Applications (9)
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DE60038572T DE60038572T2 (en) | 1999-12-10 | 2000-12-11 | USE OF A VARIETY OF RECOMBINATION CENTERS WITH UNIQUE SPECIFICITY IN RECOMBINATIVE CLONING |
AU20851/01A AU785483B2 (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique specificity in recombinational cloning |
JP2001544380A JP4729222B2 (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique selectivity in recombination cloning |
DK00984184T DK1250453T3 (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique specificity in recombination cloning |
EP00984184A EP1250453B1 (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique specificity in recombinational cloning |
NZ519217A NZ519217A (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique specificity in recombinational cloning |
CA002392753A CA2392753A1 (en) | 1999-12-10 | 2000-12-11 | Use of multiple recombination sites with unique specificity in recombinational cloning |
HK04105115.5A HK1062034A1 (en) | 1999-12-10 | 2004-07-14 | Use of multiple recombination sites with unique specificity in recombinational cloning |
AU2007221891A AU2007221891A1 (en) | 1999-12-10 | 2007-10-09 | Use of multiple recombination sites with unique specificity in recombinational cloning |
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JP (1) | JP4729222B2 (en) |
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Also Published As
Publication number | Publication date |
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CN1468317A (en) | 2004-01-14 |
EP1250453B1 (en) | 2008-04-09 |
DE60038572T2 (en) | 2009-04-30 |
EP1250453A4 (en) | 2004-04-07 |
EP2210948A2 (en) | 2010-07-28 |
NZ539430A (en) | 2006-09-29 |
DE60038572D1 (en) | 2008-05-21 |
EP2210948A3 (en) | 2010-10-06 |
JP4729222B2 (en) | 2011-07-20 |
CN100587068C (en) | 2010-02-03 |
AU785483B2 (en) | 2007-09-20 |
US20020007051A1 (en) | 2002-01-17 |
US7393632B2 (en) | 2008-07-01 |
EP1978098A3 (en) | 2009-02-18 |
JP2004500061A (en) | 2004-01-08 |
WO2001042509A9 (en) | 2002-05-30 |
CN1757724B (en) | 2014-06-11 |
NZ519217A (en) | 2004-03-26 |
NZ530816A (en) | 2005-10-28 |
ATE391789T1 (en) | 2008-04-15 |
CA2392753A1 (en) | 2001-06-14 |
EP1250453A1 (en) | 2002-10-23 |
AU2085101A (en) | 2001-06-18 |
CN1757724A (en) | 2006-04-12 |
DK1250453T3 (en) | 2008-08-11 |
EP1978098A2 (en) | 2008-10-08 |
HK1062034A1 (en) | 2004-10-15 |
AU2007221891A1 (en) | 2007-11-01 |
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