WO1999004041A9 - Methods and compositions for modulating melting temperatures of nucleic acids - Google Patents
Methods and compositions for modulating melting temperatures of nucleic acidsInfo
- Publication number
- WO1999004041A9 WO1999004041A9 PCT/US1998/014772 US9814772W WO9904041A9 WO 1999004041 A9 WO1999004041 A9 WO 1999004041A9 US 9814772 W US9814772 W US 9814772W WO 9904041 A9 WO9904041 A9 WO 9904041A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- duplex
- binding ligand
- reaction mixture
- binding
- Prior art date
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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
- 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
- C12N15/63—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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6832—Enhancement of hybridisation reaction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Definitions
- SBH involves the use of multiple probes disposed in an array format to bind to a sample of a target nucleic acid which has been cleaved into smaller fragments.
- SBH has been attempted on only small DNA targets and with small probe arrays.
- Certain problems have arisen in attempts to implement SBH schemes.
- One serious difficulty is the need to correctly discriminate between target fragments that are perfectly matched to a probe sequence, and target fragments that are bound to a probe sequence despite one or more mismatched bases.
- This "mismatch discrimination" problem presents the possibility of misidentification of sequences. The problem is especially acute when attempting to differentiate between sequences which bind with significantly different binding energies.
- AT-rich sequences bind less strongly to their complementary probes than do GC-rich sequences, of the same length, to their respective complementary probes.
- it can be difficult to distinguish between perfectly-bound AT-rich sequences and partially mismatched GC- rich sequences.
- hybridization of mismatched sequences is undesirable, as it makes the unambiguous determination of the target sequence harder to achieve.
- This invention features methods of normalizing the melting temperatures of a plurality of nucleic acid duplexes.
- the invention provides a method of normalizing the melting temperatures of at least two nucleic acid duplexes.
- the method includes the steps of contacting the at least two nucleic acid duplexes with a reaction mixture comprising a nucleic acid binding ligand which preferentially binds to one of the at least two nucleic acid duplexes; such that the melting temperatures of the at least two nucleic acid duplexes are normalized.
- a plurality of nucleic acid duplexes are provided in an array, e.g., a 96 well microtiter plate or a high density nucleic acid array, e.g., "gene chip", such that modulating the stability of at least one of the nucleic acid duplexes in the array is effected by forming a reaction mixture comprising the plurality of nucleic acid duplexes and at least one base-preferring nucleic acid binding ligand.
- the nucleic acid binding ligand is a duplex-binding ligand.
- the duplex-binding ligand is distamycin.
- the reaction mixture comprises at least two nucleic acid binding ligands, and wherein each of the at least two nucleic acid binding ligands independently binds preferentially to one of the at least two nucleic acid duplexes.
- the reaction mixture comprises at least two duplex-binding ligands.
- at least one of the at least two nucleic acid binding ligands is a single-strand-binding ligand.
- the reaction mixture further comprises at least one nonspecific nucleic acid binding ligand.
- the reaction mixture further comprises a duplex denaturant, such as, e.g.. urea.
- Figures 1A-1D show theoretical melting curves of a random mixture of nucleic acid duplexes in the presence of various nucleic acid binding ligands.
- Figure 2A shows experimental melting curves of a DNA hairpin duplex in the absence and presence of Distamycin A.
- Figure 2B plots the derivative of the curve in Figure 2A.
- Figure 3 A shows experimental melting curves of a DNA hairpin duplex in the absence and presence of ethidium bromide.
- Figure 3B plots the derivative of the curves in Figure 3A.
- Figure 4 A shows experimental melting curves of a DNA hairpin duplex in the absence and presence of Distamycin A and urea.
- Figure 4B plots the derivative of the curves in Figure 4A.
- Figure 5 A shows experimental melting curves of a DNA hairpin duplex in the absence and presence of ethidium bromide, Distamycin A, and urea.
- Figure 5B plots the derivative of the curves in Figure 5 A.
- Figure 6 shows the chemical structures of certain duplex binding ligands.
- Figure 7 shows the results of titration of the hairpin/target sets with various duplex binding ligands as shown in Example 5.
- Figure 8 shows the effect of polypeptides on hybridization as set forth in Example 5.
- Figure 9 shows the effect of distamycin A on the association rates of the three sets of target/hairpin molecules, as set forth in Example 6.
- Figure 11 shows the effect of bisbenzamide on the association rates of the three sets of target/hairpin molecules, as set forth in Example 6.
- Figure 12 shows results of titration of the hairpin/target sets with various duplex denaturants, as shown in Example 7.
- Tm melting temperature
- normalizing means the process of causing the Tms of a plurality of duplexes to approach a common temperature.
- the Tms of a plurality of duplexes are said to be “normalized” if the Tms of the "normalized” duplexes are more nearly the same, in relative or absolute terms, than the Tms of the same duplexes which have not been normalized.
- Moduleating the Tms of two or more duplexes refers to increasing or decreasing the absolute or relative difference in the melting temperature between at least two duplexes.
- duplex denaturant refers to an agent that, at some concentration, can cause the denaturation, e.g., the dissociation, of nucleic acid duplexes, either in sequence-specific, base-preferring or non-sequence-specific contexts.
- Duplex denaturants include any chemical agent that, under suitable conditions, can alter the duplex-single strand equilibrium so as to favor single strand formation and disfavor duplex formation. Increased temperature (heating) can be used instead of a (chemical) duplex denaturant, although this is not preferred.
- a duplex denaturant is a chemical or biochemical reagent.
- duplex binding ligand binds to a duplex nucleic acid with a greater binding energy than the energy with which the ligand binds to either of the single- strands which make up the duplex.
- a duplex binding ligand is a chemical or biochemical reagent.
- Exemplary duplex-binding ligands include enzymes such as polymerases, ligases, and the like; intercalators; drugs such as Berenil (diminazine aceturate), b/s-benzamide, ethidium bromide, actinomycin D and distamycin A; and the like.
- Duplex-binding ligands can be identified by measuring the Tm of a duplex in the presence and the absence of a suspected duplex-binding ligand; a duplex-binding ligand at some concentration will raise the Tm of the duplex.
- Preferred duplex-binding ligands do not have an adverse effect on other components of a reaction mixture, when used in amounts sufficient to stabilize at least one duplex.
- a duplex-binding ligand should not inhibit the activity of enzymes, such as polymerase or ligase, if activity of such enzymes is desired.
- base-preferring binding ligand refers to a nucleic acid binding ligand that preferentially binds to nucleic acid sequences (or duplexes) in which one or more specified bases predominate.
- a nucleic acid binding ligand that preferentially binds to sequences rich in A or T is a base-preferring binding ligand (also referred to as an "AT-binding ligand").
- a "base-preferring binding ligand” can be, but need not be, a sequence-specific binding ligand (which is a ligand that preferentially binds to a particular sequence or sequences), nor is a sequence-specific binding ligand necessarily a base-preferring binding ligand, although it can be.
- a ligand that preferentially binds to a sequence motif of AGCT is sequence specific (for the sequence AGCT), but is not base-preferring because the base composition in the sequence is evenly distributed among A, G, C and T.
- nonspecific binding ligand refers to a nucleic acid binding ligand that does not substantially preferentially bind to nucleic acid sequences in which one or more specified bases predominate. That is, a "nonspecific binding ligand” binds to all, or a large variety of, bases or sequences approximately equally well.
- modulating the stability of nucleic acid duplexes refers to the process of changing the stability (either increasing or decreasing) of at least one duplex in a mixture of a plurality of duplexes.
- nucleic acid strand refers to a strand of DNA or RNA, or a mixed DNA-RNA strand, or nucleic acid-like compounds such as peptide nucleic acids.
- a nucleic acid strand can also include modified (e.g., chemically or biochemically modified) D A or RNA bases, of which many are known in the art.
- AT-rich means a sequence (e.g., all or part of a strand or duplex) in which greater than 50% of the nucleic acid bases are A or T.
- RNA or chimeric RNA-DNA sequences when RNA or chimeric RNA-DNA sequences are used, it will be understood that references to thymidine (T) can also apply to uridine (U), unless indicated otherwise.
- GC-rich means a sequence (e.g., all or part of a strand or duplex) in which greater than 50% of the nucleic acid bases are G or C.
- target nucleic acid sequence refers to a nucleic acid sequence which is to be detected, sequenced, immobilized, or manipulated.
- the target nucleic acid sequence can be any nucleic acid strand, as defined above, and in general will be single-stranded or will be made single-stranded by methods known in the art.
- the target nucleic acid sequence can be obtained from various sources including plasmids, viruses, bacteria, fungi, yeast, plants, and animals, including humans; or the target nucleic acid sequence can be obtained from non-natural sources.
- the target nucleic acid sequence can be obtained from various organisms or tissues, including fluids such as blood, semen, urine and the like.
- the methods and compositions of the invention can also include one or more additional binding ligands, which can be base-preferring or sequence-specific ligands, or non-specific ligands, and can bind duplexes or single strands.
- additional binding ligands can be base-preferring or sequence-specific ligands, or non-specific ligands, and can bind duplexes or single strands.
- Ligands suitable for use in the present invention are capable, in general, of binding to nucleic acid single strands and/or duplexes. In general, it is necessary to provide at least one base-preferring ligand in the reaction mixtures of the invention.
- base-preferring ligands A variety of base-preferring ligands have been described.
- the duplex-binding ligand Distamycin A has been reported to bind preferentially to AT-rich sequences.
- Other base-preferring duplex-binding ligands include certain restriction enzymes, drugs such as actinomycin D (which has a primary binding site of 5'-GC-3', and a secondary preference for GT sites), and intercalators such as ethidium bromide (as described below).
- base-preferring single strand-binding ligands can be employed in the invention.
- Base-preferring binding ligands can be identified by methods known in the art.
- the effect of a ligand on the Tm of test sequences can be used to determine whether the ligand is a base-preferring binding ligand.
- an AT-rich duplex can be melted in the absence and presence of a candidate ligand, and a GC-rich duplex similarly melted in the absence and presence of the candidate ligand.
- a base-preferring duplex-binding ligand that preferentially binds to AT-rich sequences (an "AT-duplex binding ligand”) can, at some concentration of the binding ligand, raise the Tm of the AT-rich duplex more than the GC-rich duplex.
- a base-preferring binding ligand binds at least n percent more strongly to a preferred strand or duplex than to a nonpreferred strand or duplex, where n is 10, 20, 30, 50, 80, 100, or 150.
- a preferred AT-duplex - binding ligand can bind at least n percent more strongly to an AT-rich duplex than to a GC-rich duplex.
- the relative preference of a ligand for a particular base or bases can be measured by techniques known in the art. An exemplary technique for determining binding preference of a ligand is known as "footprinting".
- the methods of the invention can also employ more than one binding ligand, provided that at least one is a base-preferring binding ligand.
- a reaction mixture comprising a base-preferring duplex-binding ligand and a base- preferring single-strand-binding ligand can be employed in the methods of the invention.
- a reaction mixture comprising, for example, a base-preferring single-strand-binding ligand and a (nonspecific) duplex denaturant, can also be employed in the invention.
- the methods of the invention are useful in a wide variety of nucleic acid hybridization experiments in which it is desirable to modulate the melting temperatures of a plurality of nucleic acid sequences.
- Illustrative examples of experiments in which the methods of the invention find use include SBH, detection of target nucleic acids (e.g., assays), and the like.
- one application of the invention may be where it is desired to characterize and/or sequence a population of single-stranded DNA target sequences, e.g., 40mers. from a mixture.
- An array comprising bound capture moieties each having determined but differing sequences, such as described in U. S. Patent No. 5,770,365, is provided.
- the array may comprise, on the one hand, a microtiter plate having 96 positions in the array, to a "gene chip” having 96,000 positions, on the other.) Aliquots of the nucleic acid mixture of interest are placed in each array position so as to contact the capture moieties in each array under conditions favorable for hybridization, and the melting temperatures are normalized as described herein. After washing the array to remove unbound or mismatched DNA (a step which may include treatment with duplex denaturant as described herein), the bound DNA segments may be detected, sequenced, immobilized, or manipulated, etc. as known in the art.
- the invention provides a method for normalizing the melting temperatures of at least two nucleic acid duplexes.
- the method includes the step of contacting the at least two nucleic acid duplexes with a reaction mixture comprising a base-preferring nucleic acid binding ligand; such that the melting temperatures of the at least two nucleic acid duplexes are normalized.
- the base- preferring nucleic acid binding ligand is a duplex-binding ligand, such as distamycin.
- the reaction mixture comprises at least two base-preferring nucleic acid binding ligands, which can be at least two duplex-binding ligands.
- the base-preferring nucleic acid binding ligand is a single-strand-binding ligand.
- the reaction mixture further comprises at least one nonspecific nucleic acid binding ligand.
- the reaction mixture further comprises a duplex denaturant, for example, urea. The melting temperatures of at least two nucleic acid duplexes can be normalized
- the amount of binding ligand necessary to effect a desired degree of normalization can be determined by titration of the binding ligand or ligands into the mixture of duplex nucleic acids and determination of the melting temperatures of the duplexes over a range of ligand concentrations. It will be appreciated that combinations of ligands can, in certain cases, provide greater normalization of melting temperatures than a single binding ligand alone. For example, addition of an AT-preferring duplex binding ligand will tend to stabilize the formation of AT-rich duplexes, and raise the melting temperature of AT-rich duplexes.
- an AT-rich duplex has a lower melting temperature (in the absence of ligands) than a GC-rich duplex of the same length
- the combination of an AT-preferring duplex binding ligand with a GC-preferring single-strand binding ligand can normalize the melting temperatures of GC-rich sequences and AT-rich sequences by acting on both types of sequence.
- the invention provides a buffer for modulating the melting temperatures (e.g., normalizing the melting temperatures) of at least one, more preferably at least two, nucleic acid duplexes.
- the buffer includes at least one base- preferring (or sequence-specific) nucleic acid binding ligand in an amount sufficient to modulate the melting temperature of at least.
- the buffer includes at least two nucleic acid binding ligands (which can be base-specific or sequence-specific).
- the buffer can also include a duplex denaturant; a single-strand binding nucleic acid binding ligand; a duplex-binding ligand; and/or a nonspecific ligand.
- kits for modulating e.g., normalizing the melting temperature of at least one, more preferably at least two, nucleic acid duplexes.
- the kit includes a container of a nucleic acid binding ligand, which can be a base-preferring or a sequence specific binding ligand.
- the kit includes at least two nucleic acid binding ligands (which can be base-specific or sequence-specific).
- the kit can also include a duplex denaturant; a single-strand binding nucleic acid binding ligand; a duplex-binding ligand; and/or a nonspecific ligand.
- the hairpins were synthesized by standard methods on a DNA synthesizer.
- the hairpins had the following structure:
- NJO indicates a 10-bp region of random sequence, which was synthesized by programming the DNA synthesizer to use all 4 bases (i.e., A, G, C, T) for these positions; N' j 0 denotes the complement of N j 0 .
- the hairpins had a 48-base pair duplex stem (a self-complementary region 48-bp in length) linked by a -T-T-T-T- loop.
- the hairpins were synthesized by synthesizing precursor molecules (made by standard phosphoramidite chemistry on an ABI 380B synthesizer) having the structure: 5*-ACGGC CTTTC TATAG (N 10 ) GAATT CGGCG TACTC GACCG GACTT TTGTC CGGTC GAG-3'
- the sample was incubated for five hours, and then the extended hairpins were separated from unreacted molecules by polyacrylamide gel electrophoresis (PAGE).
- PAGE polyacrylamide gel electrophoresis
- Other DNA polymerases such as Large Klenow fragment (New England Biolabs) can be substituted for reverse transcriptase.
- the hairpins synthesized as described above represent a statistical mixture of the 410 possible hairpins with a 10-bp random sequence within the 48-bp duplex ("stem") region, and a 4-bp loop.
- the hairpin structure was chosen for these experiments to assure unimolecular melting transitions and avoid concentration dependence of melting (see, e.g., L.A. Marky and K.J. Breslauer, Biopolymers 26:1601-1620 (1987)).
- the random mixture of duplex structures results in a melting curve that is a composite of the individual melting curves.
- Figure 1 C shows the increase in melting temperature of AT-rich ("A+T") regions upon addition of an AT-binding duplex binding ligand to the reaction mixture of Figure 1 A; melting temperatures are lower than the control ( Figure 1A due to the presence of the denaturant).
- Figure ID shows the effect of addition of a GC-specific duplex binding ligand to the reaction mixture of Figure 1C; the melting temperature of GC-rich ("G+C”) regions has increase compared to the melting curve of Figure 1C.
- Example 1 The melting experiments described in Examples 1 - 4 were conducted by monitoring the absorbance of a sample solution at 268 nm, as is known in the art. Each sample consisted of about 0.5 ⁇ M DNA hairpins (having an optical density of about 0.5) in 1 ml of buffer (10 mM cacodylate, pH 7.9 at 25°C; 2mM MgCl 2 ; 100 mM NaCl). Additives were included where indicated (urea. Distamycin A, ethidium bromide (denoted as eth Br in the Figures)).
- the solution was placed in a 1 cm pathlength semi- micro quartz cuvette and monitored by a Hewlett-Packard 8452A diode-array spectrophotometer equipped with a temperature-controlled cell holder. Temperatures were increased from 24°C to 100°C at a rate of 1°C per minute. The curves of absorbance vs. temperature were normalized to upper and lower baselines and smoothed using a digital filter. Derivative curves were used to observe the fine structure of melting transitions. The melting temperature Tm was taken as the temperature at the midpoint of the melting transition (that is, where 50% of the duplexes had melted).
- Distamycin A is a duplex-binding ligand that is a minor-groove binder with a preference for AT-rich sequences (see, e.g., F.E. Hahn in "Antibiotics", v. 3, J.W. Corcoran and F.E. Hahn, Eds., Springer-Verlag, New York (1975), pp. 79-100).
- the shape of the derivative melting curve suggests that the melting of the AT-rich sequences has been shifted to the high- temperature region, while the Tm of the GC-rich sequences has not been shifted as much.
- the melting profile of DNA hairpins in the absence (solid lines, Figures 4 A and 4B) and presence (dashed lines) of Distamycin A and urea shows the combined effects an AT-duplex binding ligand and a duplex denaturant.
- Distamycin A was present in a 1.2:1 molar ratio to DNA hairpins; urea was present at a concentration of 10% (w/v) in the melting buffer.
- Figure 2, Example 1 Compared to the melting profile of the hairpins in the presence of Distamycin A alone ( Figure 2, Example 1), the overall Tm of the duplexes has decreased; compared to the control, the Tm is almost unchanged.
- the added duplex denaturant urea lowers the overall Tm of the duplexes, as expected.
- the distribution of melting temperatures has been altered compared to the control; the melting transitions at lower temperatures (e.g., in the range between about 60 and 75°C) appear to have been shifted to higher temperature.
- ⁇ TTCATATCCTAGGTGTAGTAGTAGTGAAAAAAAAGACGTGTGAC 3 ' X denotes biotinylated dU, and the target molecules are shown in bold face.
- the gap between the duplex formed by the target and the dangling end of the capture hairpin, and the duplex stem region is designed to eliminate effects to stability due to stacking with a preformed duplex.
- the hairpin/target duplexes shown above differ in stability because of their differences in GC content.
- Set 1 is 40% GC (4/10)
- set 2 is the least stable at 10% GC (1/10)
- set 3 is most stable at 60% GC (6/10).
- the ligands used were commercially available. These are:
- Duplex binding ligands 1. Actinomycin D (Sigma A- 1410)
- the structures of ligands 1 -5 are shown in FIG. 6.
- the target molecules were labelled with P 32 following a standard kinasing protocol.
- the labelled bands were isolated from the reaction solutions by denaturing PAGE (8 M urea, 20% acrylamide). P activity was determined by scintillation counting.
- a solution of the capture hairpin at 10 pmol/50 ⁇ l in PBS 150 mM NaCl, 10 mM phosphate, pH 7.2 was prepared. 50 ⁇ l/well was loaded on streptavidin-coated microtiter plates (Boehringer-Mannheim #1645692) and allowed to incubate for 30 min at room temperature. After the incubation period, the wells were washed 6 times with PBS, and blotted on clean Kimwipes.
- PBS 150 mM NaCl, 10 mM phosphate, pH 7.2
- a cocktail of the labelled targets was prepared by adding a sufficient amount of each target to the hybridization buffer to give a final concentration of -20,000 cpm/target/50 ⁇ l.
- the final composition of the hybridization buffer is IM NaCl, 10 mM phosphate, pH 7.2, and the specified concentration of the ligand.
- 50 ⁇ l of the target cocktail was loaded into each well, and the plate was incubated for the specified amount of time. After incubation, each reaction mixture was quantitatively transferred to a 0.2 ml tube (Costar 6547). The wells were washed once with 100 ⁇ l of hybridization buffer (without ligand) and the wash added to the tube. The tubes were sealed, and the activity was measured by Cerenkov counting.
- Actinomycin D acted as a single strand binder (i.e., denaturant) for the 60% GC and 10% GC content molecules. Increasing the concentration decreased the amount of target bound for these two sets of molecules. The fraction of target bound was nearly negligible at > 0.063 mM actinomycin D. Some normalization can be seen. It is believed from this data (without wishing to be bound by this theory) that actinomycin D serves to stabilize already-formed duplexes rather than promoting, by itself, duplex formation. As such, actinomycin D may be useful in conjunction with other duplex binding ligands such as disclosed elsewhere herein.
- Distamycin A is seen to stabilize duplex formation and normalize well. Even at the lowest drug concentration (0.001 mM), binding was at near completion for both the 10% GC and 40% GC.
- Z./_-benzimide showed an effect similar to distamycin A. All three sets of duplexes were stabilized, and at > 0.063. the hybridization is near completion. The 10% GC molecule responded to bw-benzimide even at the lowest concentration of the drug, while the 40% GC had a slower response, having a more gradual increase in its stability. Ethidium bromide showed a more gradual effect on stability of the 10% GC and
- Figure 8 shows the effect of two polypeptides on hybridization.
- the polypeptides were titrated into the hybridization reaction by 4-fold increments, from 0- 25 mg/ml.
- Poly(L-lysine-phenylalanine) affects the 10% GC molecule more than the 40% GC molecule.
- the fraction of the 10% GC target bound to the capture hairpin is equal to that of the 60% GC target, while fraction of the 40% GC target bound increased only to -0.6.
- both the 10% GC and 40% GC targets showed a decrease in the amount bound.
- Poly(L-arginine) normalized the binding of both the 10% GC and 40% GC in a similar way. The results showed an increase in the binding of these two targets at > 0.024 mg/ml, and at 0.4 mg/ml, the fraction bound was nearly equal to that of the 60% GC target.
- Figure 11 shows the effect of bisbenzimide on the 60% GC and 10% GC molecules.
- the "no ligand" experiment is the same as the one shown in Figure 10. With bisbenzimide, both sets were pulled up, with similar binding profiles. Binding was at -90% for both molecules at 40 minutes.
- Formamide showed a similar effect on the 40% GC and 10% GC target molecules. Binding of the two molecules went down to -10% at 20% (v/v) formamide, and at 30% or more formamide, binding was insignificant. The 60% GC target was affected more gradually, with binding reduced to -50% at 30% (v/v) formamide, with almost no binding at > 40% formamide.
- SSB had no effect on the binding of the 60% GC target under the above hybridization conditions.
- the effect on the 40% GC and 10% GC is more gradual, with no apparent decrease in the amount of target bound up to a concentration of > 8.3 mg/ml of SSB.
- a duplex denaturant and a concentration therefor may be selected for use after hybridization and normalization to selectively remove mismatched hybridizations.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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JP2000503246A JP2001510057A (en) | 1997-07-17 | 1998-07-16 | Methods and compositions for controlling the melting temperature of nucleic acids |
EP98936877A EP1007736A4 (en) | 1997-07-17 | 1998-07-16 | Methods and compositions for modulating melting temperatures of nucleic acids |
AU85728/98A AU8572898A (en) | 1997-07-17 | 1998-07-16 | Methods and compositions for modulating melting temperatures of nucleic acids |
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US5284597P | 1997-07-17 | 1997-07-17 | |
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WO1999004041A1 WO1999004041A1 (en) | 1999-01-28 |
WO1999004041A9 true WO1999004041A9 (en) | 1999-04-15 |
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PCT/US1998/014772 WO1999004041A1 (en) | 1997-07-17 | 1998-07-16 | Methods and compositions for modulating melting temperatures of nucleic acids |
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EP (1) | EP1007736A4 (en) |
JP (1) | JP2001510057A (en) |
AU (1) | AU8572898A (en) |
WO (1) | WO1999004041A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2016170147A1 (en) * | 2015-04-22 | 2016-10-27 | Qiagen Gmbh | Efficiency improving ligation methods |
US9554742B2 (en) | 2009-07-20 | 2017-01-31 | Optiscan Biomedical Corporation | Fluid analysis system |
Families Citing this family (22)
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US7244570B2 (en) * | 1998-08-04 | 2007-07-17 | Luminex Molecular Diagnostics, Inc. | Methods and compositions for modulating “marginally indiscriminant” hybridizations |
US7132236B2 (en) * | 2001-10-25 | 2006-11-07 | Agilent Technologies, Inc. | Composition and method for optimized hybridization using modified solutions |
WO2003091408A2 (en) * | 2002-04-26 | 2003-11-06 | University Of Utah Research Foundation | Characterization of single stranded nucleic acids by melting analysis using a double strand specific nucleic acid dye |
EP1877581B1 (en) * | 2005-05-06 | 2012-03-28 | Gen-Probe Incorporated | Methods and products for nucleic acid target capture |
US8628918B2 (en) * | 2005-05-09 | 2014-01-14 | Affymetrix, Inc. | Multiplex capture of nucleic acids |
US8632970B2 (en) | 2005-05-09 | 2014-01-21 | Affymetrix, Inc. | Multiplex capture of nucleic acids |
US7803541B2 (en) | 2005-05-12 | 2010-09-28 | Panomics, Inc. | Multiplex branched-chain DNA assays |
CN104673903B (en) * | 2005-06-20 | 2018-11-13 | 领先细胞医疗诊断有限公司 | The method for detecting the nucleic acid in individual cells and identifying rare cells in heterogeneous maxicell group |
WO2007044427A2 (en) * | 2005-10-05 | 2007-04-19 | Panomics, Inc. | Detection of nucleic acids from whole blood |
GB0619325D0 (en) * | 2006-09-30 | 2006-11-08 | Univ Strathclyde | New compounds |
US7807372B2 (en) * | 2007-06-04 | 2010-10-05 | Northwestern University | Screening sequence selectivity of oligonucleotide-binding molecules using nanoparticle based colorimetric assay |
EP2318552B1 (en) | 2008-09-05 | 2016-11-23 | TOMA Biosciences, Inc. | Methods for stratifying and annotating cancer drug treatment options |
WO2011011462A1 (en) | 2009-07-20 | 2011-01-27 | Optiscan Biomedical Corporation | Adjustable connector and dead space reduction |
ES2648564T3 (en) | 2010-10-21 | 2018-01-04 | Advanced Cell Diagnostics, Inc. | Ultrasensitive method for in situ detection of nucleic acids |
US20160024563A1 (en) * | 2013-04-05 | 2016-01-28 | Qiagen Gmbh | Method for performing a melting curve analysis |
ES2893100T3 (en) | 2015-10-12 | 2022-02-08 | Advanced Cell Diagnostics Inc | In situ detection of nucleotide variants in samples with a high level of noise, and related compositions and methods |
WO2018047148A1 (en) | 2016-09-12 | 2018-03-15 | Novartis Ag | Compounds for the inhibition of mirna |
WO2018119399A1 (en) | 2016-12-23 | 2018-06-28 | Grail, Inc. | Methods for high efficiency library preparation using double-stranded adapters |
US11274344B2 (en) | 2017-03-30 | 2022-03-15 | Grail, Inc. | Enhanced ligation in sequencing library preparation |
US11118222B2 (en) | 2017-03-31 | 2021-09-14 | Grail, Inc. | Higher target capture efficiency using probe extension |
GB202114032D0 (en) | 2021-09-30 | 2021-11-17 | Univ Strathclyde | Antivirals |
GB202206931D0 (en) | 2022-05-12 | 2022-06-29 | Univ Strathclyde | Compounds |
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US4996143A (en) * | 1985-12-23 | 1991-02-26 | Syngene, Inc. | Fluorescent stokes shift probes for polynucleotide hybridization |
US5633129A (en) * | 1989-07-13 | 1997-05-27 | Massachusetts Institute Of Technology | Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices |
US5795714A (en) * | 1992-11-06 | 1998-08-18 | Trustees Of Boston University | Method for replicating an array of nucleic acid probes |
AU7208394A (en) * | 1993-06-17 | 1995-01-17 | Research Foundation Of The State University Of New York, The | Improved nucleic acid reactions |
US5446137B1 (en) | 1993-12-09 | 1998-10-06 | Behringwerke Ag | Oligonucleotides containing 4'-substituted nucleotides |
-
1998
- 1998-07-16 EP EP98936877A patent/EP1007736A4/en not_active Withdrawn
- 1998-07-16 WO PCT/US1998/014772 patent/WO1999004041A1/en not_active Application Discontinuation
- 1998-07-16 JP JP2000503246A patent/JP2001510057A/en active Pending
- 1998-07-16 US US09/116,393 patent/US6221589B1/en not_active Expired - Fee Related
- 1998-07-16 AU AU85728/98A patent/AU8572898A/en not_active Abandoned
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2001
- 2001-03-08 US US09/802,282 patent/US20020009733A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9554742B2 (en) | 2009-07-20 | 2017-01-31 | Optiscan Biomedical Corporation | Fluid analysis system |
WO2016170147A1 (en) * | 2015-04-22 | 2016-10-27 | Qiagen Gmbh | Efficiency improving ligation methods |
Also Published As
Publication number | Publication date |
---|---|
JP2001510057A (en) | 2001-07-31 |
EP1007736A1 (en) | 2000-06-14 |
AU8572898A (en) | 1999-02-10 |
WO1999004041A1 (en) | 1999-01-28 |
US20020009733A1 (en) | 2002-01-24 |
EP1007736A4 (en) | 2004-05-12 |
US6221589B1 (en) | 2001-04-24 |
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