WO2001009385A2 - Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids - Google Patents
Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids Download PDFInfo
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- WO2001009385A2 WO2001009385A2 PCT/US2000/020571 US0020571W WO0109385A2 WO 2001009385 A2 WO2001009385 A2 WO 2001009385A2 US 0020571 W US0020571 W US 0020571W WO 0109385 A2 WO0109385 A2 WO 0109385A2
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- 0 COP(*OP(O)(OCCOc1ccc(C=O)cc1)=O)([O-])=O Chemical compound COP(*OP(O)(OCCOc1ccc(C=O)cc1)=O)([O-])=O 0.000 description 1
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Nc1ccccc1 Chemical compound Nc1ccccc1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 1
- HUMNYLRZRPPJDN-UHFFFAOYSA-N O=Cc1ccccc1 Chemical compound O=Cc1ccccc1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
- C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/11—Compounds covalently bound to a solid support
Definitions
- the present invention generally relates to the chemistry of the attachment of oligonucleotides to solid supports. More particularly the present invention relates to linking oligonucleotides to solid supports through a Schiff base type covalent linkage for capture and detection of single- and double stranded DNA and RNA targets. Background of the Invention The detection and quantification of very small quantities of nucleic acids plays an important role in the biological, forensic and medical sciences.
- nucleic acids in samples are detected by hybridization to a complementary oligonucleotide containing more than 8 contiguous nucleotides.
- a complementary oligonucleotide typically either the target or the capturing oligonucleotide contains a signal generating label, such as a radioactive-, fluorescent-, chemiluminescent- moiety or an enzyme (such as horseradish peroxidase) that through its catalytic activity yields a detectable product.
- a signal generating label such as a radioactive-, fluorescent-, chemiluminescent- moiety or an enzyme (such as horseradish peroxidase) that through its catalytic activity yields a detectable product.
- an oligonucleotide is immobilized to a solid support and is used to capture a target. The captured target is detected by hybridization with a second labeled oligonucleotide, that has a different sequence than the capturing oligonucleotide.
- solid supports suitable for immobilizing oligonucleotides include nylon, nitrocelluose, activated agarose, diazotized cellulose, latex particles, plastic, polystyrene, glass and polymer coated surfaces. These solid supports are used in many formats such as membranes, microtiter plates, beads, probes, dipsticks etc. A wide variety of chemical procedures are known to covalently link oligonucleotides directly or through a linker to these solid supports. Of particular interest as background to the present invention is the use of glass and nylon surfaces in the preparation of DNA microarrays which have been described in recent years (Ramsay, Nat. Biotechnol., 16: 40-4 (1998)).
- Suitable nucleophilic groups or moieties include hydroxyl, sulfhydryl, amino and activated carboxyl groups, while the groups capable of reacting with these and other nucleophiles (reactive groups) include dichlorotriazinyl, alkylepoxy, maleimido, bromoacetyl goups and others.
- Chemical procedures to introduce the nucleophilic or the reactive groups on to solid support are known in the art, they include procedures to activate nylon (US 5,514,785), glass (Rodgers et al, Anal.
- the diol was oxidized to the aldehyde stage that was subsequently reacted with hydrazide latex beads to form Schiff base linkages that were reduced with sodium cyanoborohydride.
- Another article of special interest as background to the present invention is by Tsarev et al. (Biorg.Khim., 16: 765-79 (1990)) that describes coupling of an aromatic aldehyde to the 5' phosphate of an oligonucleotide through alkylation.
- the product was used to probe the enzyme-T7A2 promoter complex.
- glass surfaces are activated by the introduction of amino-, sulfhydryl-, carboxyl- or epoxyl- groups to the glass using the appropriate siloxane reagent.
- immobilization of oligonucleotide arrays on glass supports has been described: by Guo et al, Nuc. Acid Res., 22: 5456- 5465 (1994) using 1,4-phenylene diisothiocyanate; by Joos et al, Anal. Biochem., 247: 96-101 (1997) using succinic anhydride and carbodiimide coupling; and by Beatti, et al, Mol.
- a Schiff base type covalent linkage is formed between a group containing an NH 2 moiety and an aromatic aldehyde or ketone to covalently link an oligonucleotide (ODN) to a solid support.
- ODN oligonucleotide
- the Schiff base type linkage is between the solid support and either the 3', or 5' end of the ODN, or between the solid support and one or more intermediate nucleotides in the ODN.
- the Schiff base type linkage is located in a combination of these sites.
- the Schiff base type covalent linkage may be situated not directly on the solid support or the ODN but on linking groups (linkers) which are themselves covalently attached to the solid support and to the ODN, respectively.
- the solid support or the ODN or both may include a linking group that includes the -NH 2 or aromatic aldehyde group which forms the Schiff base type covalent bond to join the ODN to the solid support.
- the Schiff base type covalent bond is formed between a semicarbazide group or moiety of the formula R'-NH-CO-NH-NH 2 , and the aromatic aldehyde moiety of the formula R"-Q-CHO, preferably a benzaldehyde moiety, where the group R' designates either the solid support or the ODN residue including any linker group attached to the solid support or ODN, and where the R" designates the other of said solid support or ODN residues including any linker group attached to them.
- the symbol Q in this formula designates an aromatic ring or a heteroaromatic ring which may have up to three heteroatoms independently selected from N, O and S, and where the aromatic or heteroaromatic ring may itself be substituted with alkyl, alkoxy or halogen groups where the alkyl or alkoxy group preferably has 1 to 6 carbons.
- the semicarbazide moiety is attached to a glass surface, and the benzaldehyde moiety is attached with a linker to the 3', or to the 5' end of the ODN, or to one or more nucleotides situated internally in the ODN.
- the synthetic methodologies to prepare the semicarbazide modified solid support surface and the aromatic aldehyde coupled ODNs comprise still further aspects of the present invention.
- Advantages of the solid support ODN conjugates linked together with the above-summarized Schiff base type linkages including an aromatic aldehyde or ketone, and particularly with semicarbazone linkages include (a) their ability to be formed below pH 7, (b) stability of the Schiff base- with- aromatic-aldehyde bonds and particularly of the semicarbazone-formed-with- an-aromatic-aldehyde bonds, (c) ability to attach a high percentage (typically more than 60 %, and preferably about 90 %, even more preferably 95 % or more) of the ODN to the semicarbazide moiety containing solid support, and (d) obtaining high coupling densities (preferably of about IO 4 oligonucleotides/ ⁇ m 2 and most preferably about 10 5 oligonucleotides/ ⁇ m 2 ) on unit surface of the solid support.
- Another aspect of the present invention is a general method for the isolation of single stranded DNA in a process where an aldehyde-labeled primer is used and an amplicon is immobilized on a semicarbazide containing solid support.
- the oligonucleotides linked to the solid surface with the semicarbazone bonds also contain one or more appropriately attached minor groove binder moieties, a fluorescent generating moiety and a fluorescent quencher.
- This conjugate is designed such that during amplification reactions, with a perfect complementary target, the quencher molecules are cleaved during amplification by the 5'-nuclease activity or a polymerase (as described in United States Patent No. 5,210,015 and in Witter et al, Biotechniques 22: 130-138 (1997)) resulting in a fluorescent immobilized oligonucleotide. Mismatched targets are not amplified and no fluorescent signal is generated.
- the specification of United States Patent No. 5,210,015 and the publication Witter et al, Biotechniques 22: 130-138 (1997) are expressly incorporated herein by reference.
- non-specific adsorption of the negatively charged nucleic acids to the semicarbazone or other Schiff base-modified glass surface can be largely eliminated by converting the unreacted NH 2 groups (preferably semicarbazide -R'-NH-CO-NH-NH 2 groups) into a moiety containing an anione. This is accomplished by reacting the solid support ODN conjugate with a reagent that introduces an anionic group, for example by reacting the conjugate with 4- formyl-l ,3-benzenedisulfonate group.
- unreacted silanol functions on the solid support preferably glass surface are end-capped with a hydrophobic siloxane to increase stability of the immobilized oligonucleotides.
- a hydrophobic siloxane to increase stability of the immobilized oligonucleotides.
- the semicarbazone linkages formed with the aromatic aldehyde moiety and linking the oligonucleotide with the solid support can be reduced to provide still stable solid-support-ODN conjugates.
- an ODN containing cytidine is immobilized on a solid support containing semicarbazide groups by bisulfite catalyzed covalent attachment through the cytidine nucleotides of the ODN.
- the present invention is primarily used at present for the capture and detection of nucleic acids using oligonucleotides attached to glass surfaces with the Schiff base type, (preferably semicarbazone) bonds, and more particularly for the capture and detection of PCR generated nucleic acid sequence in array format, although the use of the invention is not limited in this manner.
- the oligonucleotides immobilized on solid support in accordance with the present invention exhibit superior direct capture ability for complementary oligonucleotide, DNA and RNA sequences.
- Figure 1 is a graph showing in three dimensions the optimization of oligonucleotide attachment via semicarbazone bonds to a glass surface as a function of concentration of the oligonucleotide and pH of the medium.
- Figure 2 is a graph showing the attachment of oligonucleotide to the glass surface as expressed in units of fmol/spot as a function of time.
- Figure 3 is a graph showing the efficiency of hybridization and efficiency of oligonucleotide attachment as a function of oligonucleotide concentration applied on each spot.
- Figure 4 is the depiction of a hybridization of macroarray consisting of six ODN probes to eight different 30-mer ODN targets, the sequences of which are disclosed in Table 1, wherein each oligonucleotide is spotted in triplicate giving an array of 3x6 spots and wherein the target sequences 1 and 8 correspond to X and Y copy of the amelogenin gene and wherein all other target sequences contain nucleotide substitutions at positions indicated in bold in Table 1 and wherein match or mismatch of the base pairs formed between each probe and the target are indicated at the bottom of each ODN triplicate.
- Figure 5 is a depiction of a hybridization of the same macroarray of six ODN probes shown in Figure 4 to single stranded 235-mer PCR products generated from female or male human genomic DNAs and to 132-mer product representing isolated male copy of amelogenin gene fragment, and wherein the PCR product generated from male DNA sample represents a heterozygous equimolar mixture of female and male copies of the gene fragment.
- Figure 6 is a schematic depiction of a solid support tethered 5' nuclease assay.
- one of the solid support or the oligonucleotide (ODN) contains a nucleophilic amino group while the other contains an aromatic or heteroaromatic aldehyde or ketone capable of reacting with the nucleophilic amino group to form a Schiff base-type covalent linkage that attaches the ODN to the solid support, in a reasonably fast, high yield reaction resulting in high concentrations of the ODN per unit surface of the solid support, bound thereto by a stable covalent bond.
- the nucleophilic amino group preferably and ideally has a pKa less than 7.0
- the nucleophilic amino (NH 2 ) group is covalently linked to the solid support while the aromatic aldehyde or ketone (preferably aldehyde) is linked to the ODN.
- the solid supports used in the preferred embodiments of the invention contain the nucleophilic NH 2 group, as a primary amine (R' ⁇ NH 2 ), or as a hydrazinyl, (R'-NH-NH 2 ), oxyamino (0-NH 2 ) , or semicarbazido (R'-NH-CO- NH-NH 2 ) group.
- R' simply denotes the rest of the solid support, including a possible linking group or linker).
- the solid support in accordance with the present invention includes a semicarbazido group attached to the matrix of the solid support with a linker containing more than one atom and less than 30 atoms.
- These amino (NH 2 ) group containing moieties can be introduced on to the solid support or surface by methods known in the art. Among the several types of solid supports available in the art glass is most preferred.
- the glass surface contains the nucleophilic amino (NH 2 ) group, which, as noted above, may be primary amino hydrazinyl, oxyamino, or a semicarbazido group, linked to the glass surface with a linker containing more than one atom and less than 30 atoms. Most preferably a semicarbazido group is linked to the glass surface with the linker.
- the semicarbazido group has a pKa less than 7.0.
- the semicarbazido and other amino (NH 2 ) groups can be introduced on to the glass surface by methods known in the art, involving a reaction with an appropriate trialkyloxysilane.
- the semicarbazido group is introduced to the glass surface with a semicarbazide containing trialkyloxysilane, as is shown in Reaction Scheme 1.
- R represents an alkyl group of 1 to 10 carbons, although one of more of the R, groups can also be phenyl.
- R j is ethyl
- n is an integer, preferably having the values of 0 to 30, even more preferably 0 to 10.
- a trialkoxy siloxane compound (Formula 1) having an isocyano group attached by an alkyl chain is reacted with hydrazine to provide a trialkoxysilane including a semicarbazide (Formula 2), which is thereafter reacted with the glass surface to provide a glass surface (solid support) having a semicarbazide groups attached through the linker (CH 2 ) B (Formula 3).
- a trialkoxy siloxane compound (Formula 1) having an isocyano group attached by an alkyl chain is reacted with hydrazine to provide a trialkoxysilane including a semicarbazide (Formula 2), which is thereafter reacted with the glass surface to provide a glass surface (solid support) having a semicarbazide groups attached through the linker (CH 2 ) B (Formula 3).
- an aromatic or heteroaromatic aldehyde is covalently linked to the oligonucleotide (ODN), so as to enable the ODN to react with the nucleophilic NH 2 (preferably semicarbazide) groups linked to the solid support.
- ODN oligonucleotide
- nucleophilic NH 2 preferably semicarbazide
- a novel aspect or feature of the present invention to provide a phosphoramidite reagent that includes a protected aromatic aldehyde and which can be used for the introduction of the aldehyde group into an ODN during standard automated oligonucleotide synthesis.
- the aromatic aldehyde group or moiety is the "benzaldehyde” moiety having a linker designated "R x " attached to the phenyl ring, as illustrated in Reaction Scheme 2 by Formula 4.
- R x represents a chain of atoms, which may include a ring, and which may have the overall length of 2 to 150 atoms.
- Synthetic methods to construct R x are known in the art and are described, for example, in United States Patent No. 5,849,482 in connection with the description of synthesizing linker arms.
- R 2 represents an alkyl group of 1 to 6 carbons, an acyl group of one to 6 carbons, or the two R 2 groups together form a carbocyclic ring of 2 - 4 carbons (as in a cyclic acetal, for example in a cyclic acetal formed with ethylene glycol).
- the protected aldehyde of Formula 5 is then converted into a phosphoramidite reagent of Formula 6, as is shown in the reaction scheme. Detailed experimental conditions for this conversion are described for an example in the experimental section.
- the phosphoramidite reagent of Formula 6 is then used later to introduce the protected aldehyde into an oligonucleotide (as described below.
- the protected aromatic aldehyde can also be attached to a primary or secondary amino group that is itself attached to the 5' or to the 3' end of an ODN, or to a primary or secondary amino group that is attached to an internal nucleotide in the ODN.
- Amino-tailed ODNs can be prepared in accordance with the state-of-the-art, and are described for example in United States Patent No. 5,512,667 the specification of which is incorporated herein by reference.
- a reagent that is suitable for attaching a protected aromatic aldehyde to said amino groups at either tail end of the ODN or to one or more internal nucleotides is shown in Formula 7.
- R 2 and R x are defined as in connection with Formula s.
- Y is a reactive group (capable of reacting with a nucleophilic amine), such as a carbonate, isocyanate, isothiocyanate, mono or di-substituted pyridine, aziridine, CO-X, S0 2 -X (X is halogen), mochlorotriazine, dichlorotriazine, hydroxysulfosuccinimide ester, hydroxysuccinimide ester, azodonitrophenyl or azido group.
- a nucleophilic amine such as a carbonate, isocyanate, isothiocyanate, mono or di-substituted pyridine, aziridine, CO-X, S0 2 -X (X is halogen), mochlorotriazine, dichlorotriazine, hydroxysulfosuccinimide ester, hydroxysuccinimide ester,
- an appropriately acitivated 3-( ⁇ , ⁇ -dimethoxytolu-4-yl)propionic acid derivative can be coupled to 5-(3-aminopropyl) uridine nucleotide incorporated in the ODN as an internal base.
- Reaction Scheme 3 discloses an actual example for the synthesis of a controlled pore glass reagent 12 suitable for the synthesis of 3'- aromatic-aldehyde-tailed oligonucleotides.
- the numbers given to actual compounds are to be distinguished from numbers given to general formulas.
- the compound designated "6" in Reaction Scheme 3 is to be distinguished from Formula 6 in Reaction Scheme 2.
- a detailed description of the exemplary reaction conditions leading to the protected aldehyde function attached to a controlled pore glass support designated 12 in the scheme, is provided in the experimental section.
- the CPG support 12 includes a dimethoxytriphenylmethyl (DMT) protecting group on a primary hydroxyl function.
- DMT dimethoxytriphenylmethyl
- Reaction Scheme 3 also discloses an exemplary synthetic route to provide a phosphoramidite reagent 10, where the aldehyde function is protected as the di-acetate.
- the phosphoramidite reagent 10 can be used in accordance with the state of the art for synthesizing ODNs where the aromatic aldehyde function is at the 5' end of the ODN.
- the experimental section describes the conditions utilized for purification and deprotection of the diacetal and diacetate derivatives used in onnection with this invention.
- the presence of an aldehyde group in the oligonucleotide that was synthesized utilizing the aldehyde containing support and/or the phosphoramidite reagent 10 can be confirmed with a reaction with 2,4-dinitrophenylhydrazine, followed by reversed phase HPLC analysis. This technique clearly distinguishes the resulting hydrazone-ODN from starting aldehyde -ODN.
- the aldehyde ODNs prepared in accordance with the present invention showed no noticeable change in reactivity when stored at -20°C for months.
- the nucleophilic amino groups on the solid glass surface are reacted with the aldehyde groups attached to the 3'- or 5'- and of the ODN or to an internal base.
- the aromatic aldehyde is attached to the solid support (glass surface) and the amino group (preferably semicarbazide) is attached to the ODN.
- the coupling reactions are performed at pH's between 2 and 7, preferably at pH 6 and most preferably at pH 5. It has been found that, except for the pH the reaction conditions are not critical for the reaction.
- Q is an aromatic ring which may be carbocyclic and may be a condensed ring structure such as naphthalene, dihydro or tetrahydronaphthalene, or a heteroaromatic ring that may be 5 or 6 membered (e. g.
- T represents a valence bond or a linker like V.
- T has a carbon atom adjacent to X.
- An alternative exemplary preferred embodiment where an aldehyde modified solid support is coupled to an ODN containing a nucleophilic amino group at 3'-, 5'- or an internal base is shown by Formula 9.
- R 3 is H, C,-C 6 alkyl or C 3 -C 6 cycloalkyl
- Q is an aromatic ring which may be carbocyclic and may be a condensed ring structure such as naphthalene, dihydro or tetrahydronaphthalene, or a heteroaromatic ring that may be 5 or 6 membered (e. g.
- Reaction Scheme 4 discloses the formation of an ODN ⁇ to-glass conjugate linked with semicarbazone bonds in accordance with the presently most preferred embodiment of the invention.
- the oligonucleotide is attached to the solid support through more than one type of aromatic aldehyde containing moiety introduced at either the 3', 5' or at internal nucleotides. It is also within the scope of the present invention to immobilize a long chain DNA to a solid support that contains a semicarbazide moiety as disclosed above, with bisulfite catalyzed covalent attachment through cytidine residues, in analogy to the reaction described by Hayatsu in Biochem., 15: 2677-2682 (1976), incorporated herein by reference. Stability of the semicarbazone linkage under PCR assay conditions were determined using a model compound shown below:
- the semicarbazone conjugate (Formula 10) was treated in a PCR buffer at 95°C for 30 minutes and analyzed by reversed phase HPLC chromatography. Comparison of the treated semicarbazone conjugate with the starting material showed little or no degradation.
- a solid surface linked oligonucleotides also contain appropriately held, minor grove binder, fluorescent generating moiety and a fluorescent quencher.
- This conjugate is designed such that during amplification reactions with a perfect complementary target the quencher molecules are cleaved during amplification by the 5'-nuclease activity, in analogy to the reaction described in United States Patent No. 5,210,015 and in Witter al, Biotechniques 22: 130-138 (1997), resulting in a fluorescent immobilized oligonucleotide. Mismatched targets are not amplified and no fluorescent signal is generated. This is schematically in Figure 6.
- Materials for construction of arrays include, but are not limited to, nylon, polypropylene, nitrocellulose, glass, silicon wafers, optical fibers, copolymers and other materials suitable for construction of arrays such as are known to those of skill in the art.
- Endcapping of unreacted groups on the solid surface After the covalent attachment of the oligonucleotide the solid support via the semicarbazone linkage, the unreacted amino groups on the surface are treated with anion generating reagents aiding to limit non-specific primer and amplicon background. This is achieved by treatment of the solid surface with appropriate aromatic aldehydes (Formula 11).
- oligonucleotide when a semicarbazide-labeled oligonucleotide is coupled to aromatic aldehyde containing solid support, the unreacted aldehyde groups are reacted with anion generating reagents (Formula 12) ,where Rg and R 7 are independently H-, -COO " or -S0 3 " . Unreacted silanol groups can also be modified to further enhance surface characteristics.
- the appropriate silanes are commercially available (UCT, Bristol, PA).
- the oligonucleotide loadings on the solid surface were determined by the use of 5 '-or 3 '-aldehyde-modified oligonucleotides 32 P-labeled at opposing ends using the appropriate nucleotide triphosphate 32 P-labeled and either terminal deoxynucleotidyl transferase or T4 polinuclotide kinase.
- the 32 P-labeled oligonucleotide was reacted directly with the semicarbazide modified glass surface as small spots approximately 1.5 mm in diameter and the excess semicarbazide groups where capped by reaction with 4-formyl- 1 ,3-benzenedisulfonic acid.
- Covalently bound oligonucleotide was quantified with a phosphor imager using a appropriate standard curve. Maximum attachment was achieved in about one hour at a surface density of about 10 5 oligonucleotide molecules/ ⁇ m 2 . Reaction with oligonucleotide concentrations greater than 15 ⁇ M (>15 ⁇ M) yielded maximum immobilization on the glass surface. The hybridization potential of the oligonucleotides immobilized via a semicarbazone linker to the solid support, was tested by direct capture of a complementary 32 P-labeled oligonucleotide.
- oligonucleotide Arrays In another embodiment of the present invention, immobilized oligonucleotides are used in procedures which utilize arrays of oligonucleotides, such as sequencing by hybridization and array-based analysis of gene expression. In sequencing by hybridization, an ordered array of oligonucleotides of different known sequences is used as a platform for hybridization to one or more test polynucleotides, nucleic acids or nucleic acid populations. Determination of the oligonucleotides which are hybridized and alignment of their known sequences allows reconstruction of the sequence of the test polynucleotide.
- oligonucleotides comprising the wild-type sequence and all possible mutant sequences for a given region of a gene of interest can be placed on an array. Exposure of the array to DNA or RNA from a subject or biological specimen, under hybridization conditions, allows determination of wild-type or mutant status for the gene of interest. This is described, without using the present invention, in the prior art, for example in U.S. Patent Nos. 5,492,806; 5,525,464; 5,556,752; PCT publications WO 92/10588 and WO 96/17957, all of which are incorporated herein by reference.
- oligonucleotides or polynucleotides corresponding to different genes are arrayed on a surface, and a nucleic acid sample from a particular cell or tissue type, for example, is incubated with the array under hybridization conditions. Detection of the sites on the array at which hybridization occurs allows one to determine which oligonucleotides have hybridized, and hence which genes are active in the particular cell or tissue from which the sample was derived. Array methods can also be used for identification of mutations, where wild- type and mutant sequences are placed in an ordered array on a surface.
- Array methods can also be used in any diagnostic procedure where nucleic acid hybridization is feasible in combination with an appropriate detection system.
- the nucleic acids include DNA, RNA and sequences amplified by methods known in the art.
- oligonucleotide attachment to solid supports in accordance with the invention provides significant improvements in manufacturing and performance of the arrays.
- General The availability of oligonucleotides containing an aldehyde linker directly from the oligonucleotide synthesizer allows the immobilization of oligonucleotides to any amine containing solid support.
- oligonucleotide affinity chromatography material can be readily synthesized in accordance with the invention.
- the reaction solution was stirred for 1.0 h at 25 °C under argon and then treated with 1.0 ml of methanol and poured into 300 ml of 5% sodium bicarbonate solution.
- the mixture was extracted with ethyl acetate (300 ml) and the extract was dried over sodium sulfate and evaporated.
- the crude product was purified by silica gel chromatography eluting with a gradient of 25-50% ethyl acetate in hexane (2% triethylamine).
- Example 4 Preparation of 3-(4-Semicarbazido)propyltriethoxysilane
- Anhydrous hydrazine (3.2 ml; Aldrich, Milwaukee, WI) were dissolved in 30 ml of anhydrous acetonitrile.
- 2.5 g of isocyanatopropyltriethoxysilane (United Chemical Technologies, Bristol, PA) were added dropwise with vigorous stirring. Reaction mixture was stirred for 1 h at room temperature and the solvent was removed in vacuum.
- Example 5 Oligonucleotide Synthesis All oligonucleotides were synthesized on an ABI 392 DNA/RNA synthesizer using standard phosphoramidite chemistry. The oligonucleotides were purified by reverse-phase HPLC, and their concentrations determined by UV spectrophotometry at 260 nm (Ref) Yield was similar to that observed in normal oligonucleotide synthesis.
- Example 6 Derivatization of Glass Slides and Preparation of Oligonucleotide Arrays Preparation of Slides Glass slides were derivatized according to the standard silanization procedure described below.
- oligonucleotides were dissolved in 100 mM sodium acetate buffer (pH 5.0) at the desirable concentration and spotted manually directly on the derivatized slide as a 0.5 1 droplets following a grid pattern on a wet paper template underneath the slide. Slides were incubated at 37°C in a covered Petri dish located in a humid container for 1-5 hours. To block all unreacted semicarbazide groups on the glass surface the slides were treated with lOOmM solution of 4-formyl-l,3-benzenedisulfonic acid disodium salt in 100 mM sodium acetate buffer (pH 5.0) for 1 h at 37°C.
- FIG. 1 shows the effect of different pH's and oligonucleotide concentration on immobilization efficiency. A pH of 5 and an oligonucleotide concentration of 20 mM showed optimum immobilization on glass surfaces.
- Figure 2 shows that optimum oligonucleotide immobilization is achieved on the glass surface in about 1 hour.
- Example 7 Determination of Oligonucleotide Loading and Hybridization Efficiency
- the 5' or 3' aldehyde-modified oligonucleotides were radioactively labeled at the opposite end using [a- 32 P]ddATP (NEN, Boston, MD) and terminal deoxynucleotidyl transferase (Promega, Madison, WI), or [g- 32 P]ATP (Amersham, Arlington Heights, IL) and T4 polinucleotide kinase (NE Biolabs, Beverly, MA), respectively.
- oligonucleotide 1.2 nmol of oligonucleotide and 100 mCi of appropriate radioactive triphosphate were taken into a labeling reaction using the conditions specified by the manufacture.
- the labeled oligonucleotide was purified using NENSORBa 20 cartridge (NEN, Boston, MD). Eluate from the cartridge containing labeled oligonucleotide was dried down, dissolved in 100 ml of 100 mM sodium acetate buffer (pH 5.0) and supplemented with 9 nmol of unlabeled oligonucleotide to approximately 100 mM final concentration. Serial dilution of this stock was made using the same buffer with a two fold decrement.
- oligonucleotide solutions 0.5 1 were applied in quadruplicates to semicarbazide-derivatized glass slide and allowed to react at 37°C for 3 h.
- the glass surface was blocked and washed as described above, and bound oligonucleotide was quantified by phosphor imaging using a Bio-Rad GS-250 Molecular Imager.
- the data from the phosphor imager were converted to fmol/spot by comparing to standard curves generated from a serial dilution of known amounts of the same labeled oligonucleotide probes spotted on a microscope slide and dried down without any washing.
- an aldehyde- modified non-radiolabeled probe was immobilized on a slide as described above.
- 2.4 ' 5.0 cm cover slip was positioned over the area where the probes were spotted using 0.2 mm thick spacers made from electric tape.
- 80-100 ml of hybridization mixture (1 mM 5' 32 P-labeled complementary oligonucleotide, 5 'SSPE, 0.1% Triton X-100) was applied by capillary action between the slide and the cover slip.
- Optimum oligonucleotide target capture of about 75- 100 fmol/2 mm spot occurs.
- Example 8 Hybridization of Oligonucleotide Arrays with Short Oligonucleotide Targets or Single-stranded PCR Products Female and male human genomic DNA samples were obtained from Coriell Institute of Medical Research (NIGMS Human Genetic Mutant Cell Repository, Camden, NJ). The 235 bp amelogenine gene fragment corresponding to exon 3 was amplified by PCR using a set of primers, 5'- GCTGCACCACCAAATCATCCC-3' (SEQUENCE ID No. 15) and 5'-biotin- CTGGTGAGGCTGTGGCTGAAC-3' (SEQUENCE ID No. 16).
- the amplification reaction (100 ml) contained 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.001% gelatin, 100 ng DNA, 1 mM of each primer, 200 mM each dATP, dCTP, dTTP and dGTP, and 2.5 units of JumpStartaT ⁇ r DNA polymerase (Sigma, St. Louis, MO).
- PCR was performed in a Statagene RoboCycler Gradient 40 Temperature Cycler (Stratagene, La Jolla, CA) using 35 cycles (95°C for 1 min, 65°C for 1 min, 72°C for lmin).
- PCR products were purified by 4% non-denaturing polyacrilamide gel electrophoresis.
- One DNA strand of PCR products derived from the non- biotinylated primer was 5' end labeled using [g-32P]ATP and T4 polynucleotide kinase. This labeled strand was separated using streptavidin-coupled magnetic beads Dynabeads M-280 (Dynal, Inc., Lake Success, NY) according to manufacture's instructions.
- oligonucleotide macroarray s consisting of six oligonucleotides spotted in triplicates with 5' labeled 30-mer complementary synthetic oligonucleotide targets was accomplished the same way as it had been described in the previous section, except for the hybridization time, which was reduced to 3 h.
- Figure 4 is the depiction of a hybridization of macroarray consisting of six ODN probes to eight different 30- mer ODN targets, the sequences of which are disclosed in Table 1, wherein each oligonucleotide is spotted in triplicate giving an array of 3x6 spots and wherein the target sequences 1 and 8 correspond to X and Y copy of the amelogenin gene and wherein all other target sequences contain nucleotide substitutions at positions indicated in bold in Table 1 and wherein match or mismatch of the base pairs formed between each probe and the target are indicated at the bottom of each ODN triplicate.
- Figure 5 is a depiction of a hybridization of the same macroarray of six ODN probes shown in Figure 4 to single stranded 235-mer PCR products generated from female or male human genomic DNAs and to 132-mer product representing isolated male copy of amelogenin gene fragment, and wherein the PCR product generated from male DNA sample represents a heterozygous equimolar mixture of female and male copies of the gene fragment.
- 24 'Targets 1 through 8 represent ODNs having SEQUENCE ID Nos. 1 - 8
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP2001513640A JP2003505109A (en) | 1999-07-29 | 2000-07-28 | Attachment of oligonucleotides to solid support by Schiff base-type binding for capture and detection of nucleic acids |
AU63877/00A AU6387700A (en) | 1999-07-29 | 2000-07-28 | Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids |
CA002379828A CA2379828A1 (en) | 1999-07-29 | 2000-07-28 | Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids |
EP00950834A EP1200628A2 (en) | 1999-07-29 | 2000-07-28 | Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids |
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US09/364,320 US6339147B1 (en) | 1999-07-29 | 1999-07-29 | Attachment of oligonucleotides to solid supports through Schiff base type linkages for capture and detection of nucleic acids |
US09/364,320 | 1999-07-29 |
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WO2001009385A2 true WO2001009385A2 (en) | 2001-02-08 |
WO2001009385A3 WO2001009385A3 (en) | 2001-08-16 |
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PCT/US2000/020571 WO2001009385A2 (en) | 1999-07-29 | 2000-07-28 | Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids |
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US (3) | US6339147B1 (en) |
EP (1) | EP1200628A2 (en) |
JP (1) | JP2003505109A (en) |
AU (1) | AU6387700A (en) |
CA (1) | CA2379828A1 (en) |
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Also Published As
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AU6387700A (en) | 2001-02-19 |
WO2001009385A3 (en) | 2001-08-16 |
US6548652B2 (en) | 2003-04-15 |
US6441159B1 (en) | 2002-08-27 |
JP2003505109A (en) | 2003-02-12 |
CA2379828A1 (en) | 2001-02-08 |
US20020137045A1 (en) | 2002-09-26 |
US6339147B1 (en) | 2002-01-15 |
US20020081591A1 (en) | 2002-06-27 |
EP1200628A2 (en) | 2002-05-02 |
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