EP1155152A1 - Procedes et kits permettant de determiner la stabilite de duplex d'acide nucleique - Google Patents

Procedes et kits permettant de determiner la stabilite de duplex d'acide nucleique

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Publication number
EP1155152A1
EP1155152A1 EP99967559A EP99967559A EP1155152A1 EP 1155152 A1 EP1155152 A1 EP 1155152A1 EP 99967559 A EP99967559 A EP 99967559A EP 99967559 A EP99967559 A EP 99967559A EP 1155152 A1 EP1155152 A1 EP 1155152A1
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EP
European Patent Office
Prior art keywords
nucleic acid
duplex
strand
target
initial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99967559A
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German (de)
English (en)
Other versions
EP1155152A4 (fr
Inventor
Kenneth J. Breslauer
Craig A. Gelfand
G. Eric Plum
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Rutgers State University of New Jersey
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Rutgers State University of New Jersey
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Application filed by Rutgers State University of New Jersey filed Critical Rutgers State University of New Jersey
Publication of EP1155152A1 publication Critical patent/EP1155152A1/fr
Publication of EP1155152A4 publication Critical patent/EP1155152A4/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • nucleic acid duplexes Due to the high association constant for nucleic acid duplexes, the component concentration must be below the association constant, the components are likely to be too dilute to be detected by standard spectroscopic means. Having nucleic acids compete for duplex formation, as in the present method, creates a second equilibrium, referred to herein as a "competition" for duplex formation, that is measurable at essentially any concentration range. Thus, the concentrations can be tailored to virtually any method of detection. Further, the competition is measured directly from a single experiment rather than having to compare the results of two independently measured experiments.
  • FET provides a unique, extremely sensitive, and essentially binary means of discrimination because only a duplex with both the donor and acceptor dyes will have the spectroscopic signature of the energy transfer.
  • Competitive equilibrium assays of the present invention are more widely adaptable to a variety of nucleic acid systems than are assays that are based on changes in intrinsic spectral characteristics of the dyes.
  • the FET assay of the invention is dependent only on the presence of the two dyes and is limited only by the necessity of modifying DNA to bear the dyes and the fact that the distance between the dyes increases substantially when the initial FET duplex is disrupted. Any spectral changes that accompany the disruption of the FET duplex can be easily treated during data analysis and do not cause any significant complication for the FET assay.
  • An object of the present invention is to provide methods for screening for nucleic acid duplex stability by competitive equilibria.
  • a solution is first produced containing a known amount of an initial or reference nucleic acid duplex with a known stability.
  • the initial duplex is comprised of a first nucleic acid strand having a sequence, wholly or in part, homologous to a target strand and a second nucleic acid strand having a sequence, wholly or in part, complementary to the target strand.
  • a series of additions of target strand are then made by titrating the solution with a second solution comprising a known concentration of the target nucleic acid strand.
  • Another object of the present invention is to provide methods for determining the concentration of a target nucleic acid strand which comprises adding a known volume and concentration of an initial nucleic acid duplex with a known stability to a known volume of a solution containing a target strand.
  • a known volume of a solution of target strand can be added to a known volume of a solution containing a known concentration of an initial nucleic acid duplex with a known stability.
  • the solution is then subjected to conditions which disrupt the initial nucleic acid duplex and any duplex between the target strand and a strand of the initial nucleic acid duplex followed by conditions which promote duplex formation.
  • duplex stability of a particular defect or modification
  • the range of effects on duplex stability of a particular defect or modification can be readily determined with the methods of the invention. With this information, the most interesting duplexes can be identified for further study. Thus, large investments in time and materials will be made only for systems of real interest .
  • One of the key features of the invention is the flexibility in the nature of the test nucleic acid components that can be evaluated. In one experiment, two 13 mer DNA oligonucleotides, each bearing one of the FET fluorophores, referred to herein as donor (D) and acceptor (A) strands, form a duplex.
  • the test or target strand competitor is a third DNA oligonucleotide of the same length bearing a single damaged site at the central position.
  • nucleic acid duplex and test strand competitor.
  • the donor (D) and acceptor (A) strands need not be the same length nor must the target, whether single strand or duplex.
  • the terms "DNA”, “nucleic acid”, “oligonucleotide” and “strand” are meant to include other variations as there is no requirement for any of the three nucleic acid strands to be DNA.
  • DNA DNA
  • nucleic acid oligonucleotide
  • strand are meant to include DNA, RNA, and analogues including those comprised, in whole or in part, of modified bases and/or modified backbones such as peptido-nucleic acids (PNA) and other oligomers, incorporating modified phosphate and/or sugar moieties (e.g. PNAs, methyl phosphonates , phosphorothioates) that maintain duplex-forming ability may be used in the method of the invention.
  • PNA peptido-nucleic acids
  • sugar moieties e.g. PNAs, methyl phosphonates , phosphorothioates
  • nucleic acid amplification techniques such as polymerase chain reaction produce duplex target.
  • the technique of the present invention can be used on such targets in one of two ways.
  • FET monitored either by fluorescence of the acceptor or by quenching of the donor, is not the only usable means of monitoring the amount of the reference or donor- acceptor duplex. Any method by which the amount of reference or initial duplex can be monitored as a function of the amount of target can be used. Eximer fluorescence or other optical means can be used. In fact, nucleic acid strands of the initial duplex can be labeled with any pair of species with properties or characteristics dependent upon proximity. Examples include, but are not limited to, fluorescent dyes, antibody-antigen pairs, enzyme-inhibitor pairs and enzyme- coenzyme pairs.
  • SPR surface plasmon resonance
  • this assay can be used to evaluate nucleotide mimetics as drugs such as the anti-HIV drugs ddC and AZT; to evaluate the effects of carcinogen/chemical exposure on the stability of DNA and DNA- RNA hybrid duplexes; and for screening of various parameters for hybridization studies (e.g. temperature, buffer, sequences) .
  • the assay of the invention can be a companion technique to help improve existing hybridization techniques.
  • the method of the invention can also be used for screening, in solution, for the presence of single nucleotide polymorphisms (SNPs or "snips") which are used in pharmacogenetic research targeted at identifying the genetic basis of disease and genetic diagnosis of the potential for such disease in individuals.
  • SNPs single nucleotide polymorphisms
  • snips single nucleotide polymorphisms
  • Many companies are currently involved in developing and marketing hybridization assays for a wide variety of research and development efforts. Virtually all of the assays currently in use rely on immobilization of at least one participant in the hybridization reaction.
  • the immobilization introduces a host of complications, including non-specific interaction of any/all of the components with the immobilizing platform; possible distortion of the biochemically important equilibrium due to immobilization; the possibility that the chemical linkage of the immobilization can partially occlude the necessary interactions with the non-immobilized components; and the necessity of additional steps in the protocol for the immobilization itself prior to running the binding experiments.
  • the method of the invention being entirely in solution, eliminates the complications caused by immobilization. Further, because the method uses titration, control experiments with standardized DNA can be run frequently or in parallel with test compounds to eliminate spurious results. Such standardized DNA is a component of a kit for carrying out the method of the invention.
  • the competing equilibria which are the basis of this assay provide a greatly enhanced method for detecting differences in stability between two nearly identical duplexes. Studying the association of two strands forming one duplex and comparison of the association of two other strands in a separate experiment, as is done in current methods, requires two experiments with the inherent compounding of experimental error. The present invention advantageously requires only one.
  • the calculations used for data analysis are derived from the general equations for three component, two-equilibria systems as taught by Linn and Riggs (J. Mol. Biol. (1972), 72, 671-90) .
  • AD, AX, D, A, and X represent the donor/acceptor complex, the competitor/acceptor complex, donor strand, and acceptor strand, and the competitor or "test" strand, respectively:
  • ⁇ is the fraction of the initially observed fluorescence energy transfer at each point in the titration and is related to the relative concentrations of the donor/acceptor complex
  • Equation 8 The value of K ⁇ , will have been previously determined by independent methods such as differential scanning calorimetry and UV-absorbance melting. Alternate representations of equation 8 can be used to evaluate the free energy changes associated with the formation of the duplexes and the defects.
  • the impact of a difference between two oligonucleotides (X x & X 2 ) on duplex stability, ⁇ G°, can be evaluated by titration (in separate experiments) of the two competitors against the same reference AD duplex at the same AD concentration (Equation 12) .
  • ⁇ G ° AX values that are measured are relative to the value of K AD .
  • titration of X into the solution to a concentration of 1000 [D] t is convenient.
  • This is a rather large range and should accommodate most single base defects.
  • the range can easily be extended by performing additional titrations using less stable AD duplexes.
  • the stability of the AD duplexes can be modulated by inclusion of modified bases and/or mismatches.
  • a series of AD duplexes can therefore be designed to cover essentially any range of ⁇ G° values, in intervals of 3 to 4 kcal/mole .
  • Equation 6 can be rearranged and used to determine the concentration of a target sequence (X) , when the values of K AD and K M are known.
  • a known volume and concentration of AD duplex is added to a known volume of an X containing solution.
  • a known volume of X containing solution is added to a known volume of AD duplex containing solution of known concentration.
  • the concentration of X, [X] t can be calculated from the relative change in fluorescence, ⁇ , using the formula
  • the cooling process can be performed in steps so that values of ⁇ are collected as a function of temperature. Data at each temperature are used to produce a family of titration curves. Each curve is analyzed independently and values for K M are determined as a function of temperature.
  • Equation 16 can be used to extract enthalpy ( ⁇ H°) data.
  • a competition assay is usable over a wide range of instrumentally accessible concentrations.
  • the method of the invention provides a more reliable measurement of nucleic acid complex stability over a very wide range of free energy values because the titration depends on the difference in stability between the initial donor/acceptor-containing duplex and the resulting competitor-containing duplex and not on their absolute free energy values. Therefore, the range of accessible free energy values can be tuned by choice of the initial donor/acceptor-containing duplex. Relative free energies are usually the desired experimental result and the method of the invention provides them directly.
  • the lower detection limits of the fluorophores define the maximum difference in free energy that can be detected by the method.
  • the use of fluorophore detection provides great sensitivity.
  • the emission spectrum of the donor strand at 10 pM concentration has been visualized reliably using a photon- counting fluorometer.
  • Free energies calculated from the assay of the present invention have been demonstrated to be in agreement with those measured by extensive thermodynamic studies on individual duplexes.
  • two titrations were performed at the same D t concentration, for two starting Watson-Crick FET duplexes, designated A»T and T * A, which differ only by the central base pair, out of the 13 pairs in the duplex.
  • Competition on each duplex is from nearly the same single strand as present in the FET duplexes, except this single strand is unlabeled and has a tetrahydrofuranyl abasic "lesion" site (F) at the central base pair.
  • Free energy values measured by this method compare quite favorably to those measured by extensive differential scanning calorimetry and UV absorbance melting experiments on these 13-mer duplexes containing a single tetrahydrofuranyl abasic site (F) in the central position.
  • F tetrahydrofuranyl abasic site
  • [D] t / [X] the method of the present invention can be used in any concentration regime.
  • the appropriate concentration range is determined by the sensitivity of the detection system. Accordingly, [D] t is selected by one of skill to optimize the detection method. Due to practical limitations on the volume of titrant that can be added to the titrate, there are some practical limitations to the range of free energy values that can be covered by a single titration. However, this range is quite large.
  • a convenient limit of the ratio [D] J [X] 05 is about 0.001. This corresponds to a factor of about 0.002 in association constant or 3.7 kcal/mol in free energy. Most defects for which reliable free energy data are available fall into this range.
  • the assay may be tuned over a wide range of free energy values by introducing mismatches in the donor-acceptor reference duplex.
  • a series of three donor acceptor duplexes can cover a range of 11 kcal/mole in free energy.
  • titrations are done in parallel, use of such a family of duplexes relieves one of estimating the magnitude of K M prior to performing the titration.
  • several donor/acceptor complexes are formed simultaneously.
  • Each different donor or acceptor oligonucleotide differs slightly, while maintaining complementarity of the acceptor with the target (X) or employing an analogous system where X binds to the donor, to produce donor/acceptor pairs of differing stability.
  • donor and acceptor dyes it is possible to conduct multiple simultaneous titrations of X into a single solution. Spectroscopic discrimination of the various donor acceptor pairs provides multiple free energy determinations simultaneously.
  • the assay of the present invention can be adapted by immobilization to a variety of inert solid supports using technologies established for standard hybridization studies.
  • one of the strands of the reference duplex can be attached to the surface.
  • the reference duplex can then be formed on that surface. Exposure to target will release the unattached strand, thereby producing the signal. Because concentration cannot be defined at a surface in the same way as in solution, comparison to a standard of known stability is required for quantitative results.
  • immobilization facilitates the miniaturization and adoption of this assay to high throughput screening while providing the benefit of using FET and the competing equilibria to increase the sensitivity of measurement of differences in duplex stability.
  • Simultaneous titrations provide a number of additional advantages to this assay.
  • the range of accessible free energy is multiplied by the number of duplexes titrated simultaneously.
  • employing three simultaneous donor-acceptor duplex titrations means that the effective range of a single titration experiment becomes 9-12 kcal/mol, without expending any extra test strand.
  • the enhanced range also means that a favorable outcome is likely in a single experiment, rather than having to explore various single donor acceptor duplexes to find one which has a free energy less than 3-4 kcal/mol higher than the test duplex.
  • Multiple donor acceptor pairs are designated A-J - ⁇ , A 2 D 2 , A 3 D 3 , etc.
  • An example is provided using three AD pairs, but any number is possible. Equations accounting for the multiple simultaneous equilibria are described below.
  • a fundamentally different strategy for simultaneous monitoring of multiple donor/acceptor pairs is to use the dyes as acceptor and donor, but on different duplexes. Again, discrimination is made optically.
  • an example is provided using three donor/acceptor complexes, but the method is not limited in the number of complexes that can be employed.
  • the oligonucleotides are designed so that D A 2 D D 1; D A 3 D D 2 , and D A 4 D D 3 vary in stability systematically and so that the X strand can form duplexes with the acceptor bearing strands, namely D A 2 X, D A 3 X and D A 4 X.
  • equilibrium constants can be derived relating the concentrations of the various solution components .
  • the concentrations of the donor/acceptor complexes can be determined by measurement of the ⁇ values and knowledge of the total concentrations of the donor strands .
  • a series of different strands can be attached to a surface so as to identify a particular oligonucleotide with its location on the surface. This kind of spatial distribution of different oligonucleotides is well known.
  • the fluorophore can be attached post-synthetically or as a phosphoramidite; either method is compatible with methods for producing an oligonucleotide array on a surface.
  • a single oligonucleotide with sufficient complementarity to form reference duplexes with each of the immobilized strands can be used to form a series of reference duplexes . The surface is then exposed to a single target that simultaneously equilibrates with all of the reference duplexes.
  • SNP single nucleotide polymorphism
  • the theory behind the application of this FET assay to SNP screening is based upon two segments of DNA, the first being a "wild type" sequence (does not contain a SNP) , and the second being a variant sequence (a SNP) that, for example, may be a marker for disease or disease tendency.
  • Standardized methods abound for amplifying a small genetic sample (e.g. from a few microliters of blood) , and the result would be one amplified strand, which would become the unlabeled competitor
  • (X) strand in our FET assay When amplification of the target is performed the quantity of the target can be determined by use of labeled primers. Either fluorescently labeled, so as not to interfered with the subsequent FET measurement, or by any of the many well known methods for labeling amplification products .
  • SNP screening is merely a detection of a SNP, and not a quantitation of energetic impact per se
  • FET-based SNP screening does not even require a full titration of target (X) into the donor-acceptor duplex.
  • X target
  • Merely adding a fixed excess amount of the X strand is sufficient, in effect making a two-point titration wherein the fluorescence is measured before and after X addition.
  • the amount of X strand needed should be in the range of 10- to 100-fold excess over donor (D) .
  • D donor
  • the magnitude of ⁇ G° is not a consideration, since the fluorescence assay measures the difference in two ⁇ G° values ( ⁇ G°) directly. Mismatches of the four canonical bases will generally exhibit ⁇ G° values of about 1 or 2 kcal/mol, but this 1 to 2 kcal/mol is the actual magnitude of the measurement itself. Thus, whether the absolute values are 20 kcal/mol of duplex, as in a 10-15 base pair oligonucleotide, or 600 kcal/mol of duplex, in something perhaps as long as a gene, the assay is silent to these magnitudes, because it is reporting ⁇ G° directly.
  • the dried samples are then dissolved in H 2 0 at low temperature ( ⁇ 40°C) and floating non-soluble materials are removed.
  • the sample may be purified by reverse phase, using high performance liquid chromatography (HPLC ) and a PRP- column, equilibrated with 50 mM ammonium bicarbonate. Elution is performed by a linear (5-50%) gradient of acetonitrile in 50 mM NH 4 HC0 3 .
  • Trityl is then removed by addition of 200 ⁇ l of 80% acetic acid for one hour at room temperature and evaporating the liquid in a Speed -Vac for 2 to 3 hours until a glassy residue is observed. This step is critical to ensure elimination of any residual free amines that might interfere with the labeling reaction.
  • the trityl group is removed by addition of 80% acetic acid and incubation for one hour at room temperature, followed by freeze drying. Both the purity of the final product and the success of detritylation are monitored by analytical reverse phase HPLC. If required, additional purification of the detritylated oligonucleotide is performed by reverse phase HPLC.
  • DNA (30-40 OD-260) is dissolved in 270 ⁇ l of H 2 0 in an O-ring tube. NaHCO 3 (30 ⁇ l, 1 M) at pH 8.3 is then added. One mg of succinyl ester form of the dye is then added for each 20 OD of DNA. This can be weighed as a dry reagent into a glass vial, dissolved in 80 ⁇ l/mg of fresh DMSO, and added into the plastic tube of DNA solution. The glass vial is then rinsed with 20 ⁇ l of additional DMSO and added to the plastic tube. Alternatively, a 100 ⁇ l aliquot of dye is added. The dye and DNA are then allowed to react at 37°C or higher at least overnight.
  • the DNA is then isolated from any unreacted dye with a PD-10 Sephadex G-25 column (Pharmacia) .
  • the column is equilibrated by rinsing with at least 25 ml of H 2 0.
  • the DNA sample is diluted with H 2 0 to a final volume of 1000 ⁇ l and loaded onto the column.
  • the DNA is then washed using 1.6 ml H 2 0.
  • DNA is eluted in 600 ⁇ l fractions with H 2 0. Generally six fractions are sufficient.
  • the fractions are dried to at least % the volume and adjusted to 300 ⁇ l total volume with H 2 0.
  • Each fraction is then independently subjected to ethanol precipitation. Generally only the first three fractions will have appreciable DNA.
  • the free dye stays mostly dissolved in the ethanol.
  • the labeled DNA is further purified by HPLC using an ion exchange column (e.g. Mono Q, Pharmacia) equilibrated with 50 mM Tris HCl and 15% acetonitrile (Buffer A) and a linear gradient (0-100%) of Buffer B (i.e., Buffer A containing 1 M NaCl) .
  • Buffer A i.e., Buffer A containing 1 M NaCl
  • Absorbances at 260 nm and the wavelength corresponding to the maximum absorbance of the fluorophore are use to monitor and define labeled and unlabeled pools of oligonucleotides.
  • a desalting column can be used in place of the ethanol precipitation steps .
  • the labeled oligonucleotides may be stored as stock solutions in water and working buffer at or below -20°C for at least six months. Preferably, the samples should be stored as a lyophilized powder, for periods exceeding six months.
  • Example 2 Determination of labeled DNA concentration
  • Determination of the concentration of the labeled DNA strands in stock solutions has been performed using an average extinction coefficient of 1.1 x 10 5 M ⁇ cm "1 at 25°C.
  • the intrinsic DNA absorbance at 260 nm has been demonstrated to not be significantly altered by the presence of the conjugated dye .
  • the time drive is set to collect, using the kinetics mode, 30 or 60 seconds (at 0.1 second per reading) using 508 nm excitation and 528 nm emission. These data points are then averaged, resulting in a precise relative fluorescence intensity for each reading, and an associated standard deviation for that averaged value.
  • the fluorescence of the buffer alone is read to establish a blank for the instrument response.
  • the fluorescence of the "free" donor strand is then determined.
  • a sample of 10 nM donor strand is prepared in 250 ⁇ l total volume of buffer and fluorescence is measured.
  • An aliquot of the acceptor strand from the working stock sufficient to achieve a final concentration of 100 nM is then added to form the FET duplex and the fluorescence is determined.
  • Example 4 Titration of the competing strand
  • a working stock of the competing strand is prepared at a concentration appropriate to the expected ability/inability to compete for duplex formation with the formed donor/acceptor pair. In general, the concentration is one order of magnitude higher than the donor and acceptor working stocks for each 1 kcal/mol of free energy difference
  • concentrations expected for the competing strand.
  • concentrations can be prepared, covering a wide range of concentrations, i.e., 2 ⁇ M, 20 ⁇ M, 200 ⁇ M, etc. solutions in buffer. Titration is started with the most dilute solution and more concentrated solutions are used as necessary.
  • the competing strand is added to a final concentration of about l A of the concentration of the donor/acceptor concentration.
  • the fluorescence is then determined. If there is a significant change in the fluorescence, titration is continued with the working stock. If there is no change, titration is continued with a higher working stock concentration. Additional aliquots are added and the fluorescence determined until the fluorescence intensity recovers to at least % of the intensity measured for the free donor strand.
  • the FET data acquisition protocol was automated on an AVIV Model ATF-105 Automatic Titrating Fluorescence Spectrophotometer .
  • the customized software developed for the FET assay on this particular instrument improved the overall accuracy, precision, and data throughput compared to conventional manual titration experiments.
  • There are several key features of the automated FET assay including programmed titration of acceptor and competitor or target strands and the ability to conduct successive heating/cooling cycles of the sample solution in the cuvette. Selection of the upper temperature limit is dictated by two important considerations, namely that the parent and test duplexes are dissociated into single strands and the covalently attached fluorophores are stable at the desired upper temperature.
  • a typical experiment is initiated by placing a cuvette containing a 100 nM solution of the Oregon Green 514 labeled donor strand in the sample compartment, heating the cuvette to 75°C, maintaining the temperature at 75°C for three minutes, and cooling the sample to 20°C over an equilibration period of five minutes.
  • the experimental protocol may be simplified by loading the pre-formed reference duplex into the cuvette prior to conducting the automated competition experiment. Elimination of the forward titration increases the sample throughput by reducing overall experimental time in the FET assay by approximately 50 percent .
  • the second syringe drive is activated to dispense fixed aliquots of the unlabeled competing strand (X) d(CGCATGFGTACGC) (SEQ ID NO: 3) .
  • the sample solution containing the three strands is subjected to heating/cooling cycles after each addition in the titration experiment to facilitate annealing of the donor-acceptor and acceptor-target (AX) duplexes.
  • a sufficient excess of the target strand X (in this case approximately 100 fold) is titrated into the cuvette to ensure that at least half of the acceptor has been displaced from the reference duplex.
  • the dilution corrected relative fluorescence ( ⁇ ) is plotted as a function of the concentrations of acceptor (A) and target (X) strands.
  • K M acceptor/target association constant
  • Const xtol 0.000000000001
  • Const ftol 0.000000000000001
  • Const ntol 100
  • Const xtol 0.000000000001
  • Const ftol 0.000000000000001
  • Const ntol 100

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Abstract

Cette invention concerne des procédés et des kits simples permettant de déterminer la stabilité thermodynamique de duplex d'acide nucléique et de polymorphismes polynucléotidiques simples via des équilibres concurrentiels.
EP99967559A 1998-12-23 1999-12-23 Procedes et kits permettant de determiner la stabilite de duplex d'acide nucleique Withdrawn EP1155152A4 (fr)

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US11373198P 1998-12-23 1998-12-23
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US119909P 1999-02-12
PCT/US1999/030751 WO2000037686A1 (fr) 1998-12-23 1999-12-23 Procedes et kits permettant de determiner la stabilite de duplex d'acide nucleique

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US5925517A (en) * 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
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