WO2004104196A1 - Composition tampon - Google Patents

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Publication number
WO2004104196A1
WO2004104196A1 PCT/JP2004/007162 JP2004007162W WO2004104196A1 WO 2004104196 A1 WO2004104196 A1 WO 2004104196A1 JP 2004007162 W JP2004007162 W JP 2004007162W WO 2004104196 A1 WO2004104196 A1 WO 2004104196A1
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Prior art keywords
primer
nucleic acid
region
oligonucleotide
salt
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PCT/JP2004/007162
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English (en)
Japanese (ja)
Inventor
Joji Oshima
Ken Nemoto
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G & G Science Co., Ltd.
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Priority claimed from PCT/JP2003/006275 external-priority patent/WO2003097828A1/fr
Application filed by G & G Science Co., Ltd. filed Critical G & G Science Co., Ltd.
Priority to JP2005506389A priority Critical patent/JPWO2004104196A1/ja
Publication of WO2004104196A1 publication Critical patent/WO2004104196A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a buffer composition useful for a nucleic acid synthesis reaction.
  • the complementary strand synthesis reaction of nucleic acid using DNA synthase and RA synthase is known.
  • the c- phase capture synthesis reaction is based on the base constituting ⁇ type at the 3 and 3 ends of primers annealed to ⁇ type nucleic acid. This is a reaction for adding a base complementary to.
  • a double-stranded nucleic acid having a base sequence complementary to type I and type II is generated.
  • the complementary strand synthesis reaction has been applied to various operations for isolating and analyzing genes.
  • Gene analysis is one of the major uses of complementary strand synthesis.
  • primer annealing is required for the complementary strand synthesis reaction. In other words, unless the primer is annealed, the complementary strand synthesis reaction does not proceed.
  • a nucleic acid detection method and identification method using a complementary strand synthesis reaction product as an index have been put to practical use (Japanese Patent Publication No. 4-67957 ⁇ ).
  • a method for detecting a gene based on the PCR method is known as a method for detecting a gene having excellent sensitivity and specificity.
  • an oligonucleotide having a base sequence complementary to a base sequence to be detected is used as a primer.
  • the nucleotide sequence assumed to be detected is referred to as a target nucleotide sequence. If a nucleic acid containing the target base sequence is present in the sample, the primer annealing and the complementary strand synthesis reaction proceed. However, if no nucleic acid containing the target base sequence is present, neither anneal nor complementary strand synthesis reaction occurs. Therefore, when a PCR reaction product is detected, the presence of the target base sequence can be proved. PCR is a powerful analysis tool when the target nucleotide sequence is known.
  • the PCR method exhibits excellent sensitivity and specificity when proving the existence of a specific base sequence. This means that in order to identify similar nucleic acids by PCR, a base sequence capable of discriminating between them must be selected as a target base sequence. Therefore, in order to distinguish the differences between unknown nucleic acids and nucleic acids with extremely high similarity by PCR, the design of primers that enable such differences has been a major issue.
  • the present inventors have developed a GP method (Geno-Patern technology) as a method for solving the problem of the PCR method.
  • the GP method is a nucleic acid analysis method that can clearly distinguish differences between nucleic acids having high similarity.
  • complementary strands are synthesized for a plurality of regions of a test nucleic acid.
  • the primer and the phase-trapping synthesis reaction are designed so that each complementary strand is synthesized as a single strand as much as possible.
  • a state in which nucleic acids without complementary strands are mixed in one reaction system is created.
  • each nucleic acid Since each nucleic acid is derived from a different region, it contains a different base sequence. If any contains a partially complementary base sequence, they hybridize to each other. But because they are not perfectly complementary, hybridization is only a partial phenomenon. Similarly, if a nucleic acid has a complementary base sequence, it may hybridize to itself. In this way, a complex entangled nucleic acid aggregate is formed.
  • the aggregate in the GP method is schematically shown in FIG. Figure
  • phase capture chain is synthesized by using the primers shown in “Waveform generation primers” with a plurality of regions of the test nucleic acid as ⁇ . This synthetic product constitutes the aggregate.
  • an aggregate of nucleic acids thus constructed is to be analyzed. That is, a nucleic acid is identified by comparing the melting curves of the aggregate. Collection of nucleic acids in GP method Since the coalescence contains various combinations of double-stranded nucleic acids, multiple melting temperatures are recorded in the melting curve. If the conditions of the complementary strand synthesis reaction are the same, the pattern of the melting curve obtained from the same test nucleic acid is the same. Since the melting curve pattern is unique to the test nucleic acid, the nucleic acid can be identified by comparing the melting curves.
  • phase capture synthesis is performed for a plurality of regions of a test nucleic acid.
  • multiple primers are required. In other words, it is only necessary to design individual plies for the area to be combined. However, if ply of shorter nucleotide sequence can be used, ply may be shared among multiple regions.
  • 7 mer primer the probabilistic, can be expected to Aniru once per 6 3 8 4 bases (4 7). That is, for example, by using one kind of primer having a length of 7 mer, it is possible to obtain (total length of the test nucleic acid 163884) kinds of synthetic products.
  • the small number of primers required for the reaction is an economic advantage. In addition, if the reaction is composed of a small number of primers, it is easy to keep the conditions for annealing primers between each region constant, which should contribute to improvement of reproducibility.
  • a short ply has a low melting temperature (hereinafter abbreviated as ⁇ ), so a low annealing temperature is required.
  • Tm (° C) is derived based on the Wallace equation "2 (A + T) +4 (G + C) ⁇ (Thein and Wallace, The use of synnt hetic oligo— nucleotide as specific hybridization probes in the diagnosis of genetic disorders. In human genetic deseases: A practical approach, ed. KE Davies, pp33-50. IRL Press, Oxford, U).
  • the primer's temperature for PCR and the like is set at a temperature about 5 ° C lower than the Tm of the primer. That is, if Tm is 38 ° C, 33 ° C is Ring temperature.
  • a temperature of 60 ° C or higher is usually used for annealing.
  • the phase complementary chain synthesis reaction under a low temperature condition of 50 ° C. or less involves various obstacles.
  • the lower annealing temperature causes an increase in non-specific annealing.
  • the phenomenon of annealing not only for complementary base sequences but also for base sequences with low complementarity increases.
  • annealing is less likely to occur due to a change in the structure of the primer.
  • non-specific aggregation between primers due to a decrease in temperature or a decrease in the physical flexibility of primers is thought to hinder complementary strand synthesis.
  • primers for complementary strand synthesis usually require a length of at least about 20 bases.
  • random primers are used for complementary strand synthesis of multiple regions.
  • the random primer is an aggregate of oligonucleotides having a length of about 1 Omer.
  • the oligonucleotide constituting the random primer has an arbitrary base sequence.
  • Random primers are generally used for polymorphism analysis and probe synthesis. Phase capture synthesis reactions using random primers usually do not require specificity. Therefore, complementary chain synthesis is carried out under low stringency conditions. In other words, random primers are used to actively induce non-specific annealing.
  • the reaction conditions of the random primer cannot be applied to the GP method.
  • a technology that enables a specific complementary strand synthesis reaction under low temperature conditions is required. Disclosure of the invention
  • An object of the present invention is to provide a buffer composition useful for a complementary strand synthesis reaction under low temperature conditions.
  • Stringency is governed by salt concentration in addition to temperature conditions. Furthermore, if components that influence hybridization, such as denaturing agents, coexist in the solution, the stringency will also vary depending on these components. Therefore, it is possible to adjust the stringency to some extent by adjusting the amounts of these components while maintaining a constant temperature.
  • a decrease in salt concentration acts to increase stringency.
  • annealing at a higher salt concentration is easier than at a lower salt concentration.
  • Mg 2+ ions Mg 2+ ions
  • it is often attempted to reduce the stringency by increasing the concentration of Mg 2+ ions in PCR and other methods. If the reaction does not proceed sufficiently, dimethyl sulfoxide (MS0), polyethylene glycol, or glycerin is added to enhance the reaction.
  • MS0 dimethyl sulfoxide
  • polyethylene glycol polyethylene glycol
  • glycerin is added to enhance the reaction.
  • annealing at room temperature for example, is impossible.
  • the inventors of the present invention have tentatively studied various combinations of additives in order to find a buffer that enables sufficient complementary strand synthesis while maintaining specificity under low-temperature conditions. As a result, they have found that the above problem can be solved by adding specific salts in addition to divalent or monovalent metal salts that constitute a general buffer solution for complementary strand synthesis reaction. Furthermore, it has been confirmed that sufficient complementary strand synthesis reaction efficiency can be achieved by adding a denaturant, thereby completing the present invention. That is, the present invention relates to the use of the following buffer composition as well as the buffer composition.
  • a buffer composition for nucleic acid synthesis comprising the following composition:
  • composition according to [1] wherein the buffer is a buffer that gives a pH of 7.5-9.
  • the Mg 2+ salt is MgCl 2 .
  • composition according to [4] The composition according to [1], wherein the monovalent metal salt is one or both of KC1 and NaCl.
  • sulfate, sulfite, phosphate, and carbonate is Na 2 S0 4, Na 2 S0 3, NaH 2 P0 4, and NaHC0 any salt selected from the group consisting of 3 [ [1] The composition according to [1].
  • composition according to [6] The composition according to [1], wherein the denaturing agent is selected from the group consisting of a nonionic surfactant, an anionic surfactant, and a detergent.
  • the nonionic surfactant is selected from the group consisting of polyoxyethylene ether of glycerin ester, polyoxyethylene ether of sorbitan ester, and polyoxyethylene ether of sorbitol ester.
  • the detergent is dodecyl sulfate, lauroyl sarcosine, laurylate, [6]
  • the composition according to [6] which is any compound selected from the group consisting of mercaptoacetate and mercaptoacetate.
  • composition according to [9] The composition according to [1], wherein the nucleic acid to be synthesized is a complementary strand having a plurality of regions of a nucleic acid of type III as a type II.
  • At least one salt selected from the group consisting of 0.01 -0.1M, sulfate, sulfite, phosphate, and carbonate.
  • a stabilizing agent for oligonucleotide annealing which comprises one or both of the following components d) and e) as an active ingredient.
  • the present invention relates to a buffer composition
  • a buffer composition comprising the following composition:
  • a buffer that provides a pH suitable for a phase capture synthesis reaction by a nucleic acid synthase a buffer that provides a pH suitable for a phase capture synthesis reaction by a nucleic acid synthase
  • One 20 OmM monovalent metal salt d) 0.05-0.50 of at least one salt selected from the group consisting of sulfates, sulfites, phosphates and carbonates, and
  • an oligonucleotide refers to a molecule in which a plurality of nucleotides are linked.
  • Nucleic acid on the other hand, means a polymer of multiple nucleotides.
  • Nucleic acids include DNA and RNA.
  • Oligonucleotides can also be DNA, RA, or hybrid molecules of both.
  • the primer hybridizing to type II is particularly expressed as anneal.
  • the buffer composition of the present invention for the compositions a) to c), the same components as those constituting a reaction solution for a known complementary strand synthesis reaction can be used.
  • the buffer giving suitable pH to the complementary strand synthesis reaction by a) a nucleic acid synthetase can be used a buffer to provide a P H of neutral to weakly Al force Li.
  • the optimal pH of many commercially available DNA polymerases is 7.5-9, preferably 8-8.5. Therefore, buffers that provide this pH are useful in the present invention.
  • a Tris-HCl buffer and the like can be exemplified.
  • the concentration can be, for example, 1 to 50 mM, usually 5 to 20 mM, and preferably 8 to 15 mM.
  • concentrations of the components that make up the buffer composition refer to the final concentration unless otherwise noted.
  • Mg 2+ salts in the compositions of the present invention and Mn 2+ salt is the one, also can be utilized in combination with both.
  • MgCl 2 or MnCl 2 can be used as the Mg 2+ salt and the Mn 2+ salt.
  • These salts are usually 1-5 OmM, for example 0.5-2 OmM, preferably 0.8-1 OmM can be added.
  • a monovalent metal salt of 120 OmM is generally added to control hybridization of oligonucleotides.
  • natrium and NaCl are used as monovalent metal salts.
  • the monovalent metal salt in the composition of the present invention may be used alone or in combination of plural kinds. These salts can be added usually at a concentration of 11 to 200 mM, for example, 5 to 10 OmM, preferably 10 to 8 OmM. When a plurality of salts are combined as a monovalent metal salt, the total concentration of metal ions is adjusted so as to fall within the above concentration range.
  • compositions d) and e) are components necessary for annealing the primer under low-temperature conditions, which is the subject of the present invention.
  • either selected sulfates, sulfites, phosphates, and as the carbonate was example, if Na 2 S0 4, Na 2 S0 3, NaH 2 P0 4, and from the group consisting of NaHCO 3 Salts can be used. These salts can be used alone or in combination with different salts in the buffer composition of the present invention.
  • the concentration of these salts can be usually from 0.005 to 0.5M, for example, from 0.01 to 0.1M, and more specifically from 0.03 to 0.05M.
  • Sulfates, sulfites, phosphates, and carbonates When combining multiple types of salts, adjust so that the total concentration of salts is within these concentration ranges.
  • the denaturing agent in the present invention is a compound having an action of eliminating the higher-order structure of nucleic acid.
  • a nucleic acid may have a higher-order structure, for example, even if it is a single-stranded polynucleotide. Especially at low temperatures, it is thought to have various higher-order structures instead of a simple chain structure.
  • the denaturing agent is considered to have an effect of assisting the annealing of the oligonucleotide by eliminating the higher-order structure of the nucleic acid.
  • Nonionic surfactants are classified into ether type, ether ester type, ester type, and nitrogen-containing type.
  • nonionic surfactants particularly from non-ionic surfactants are preferred c ether ester type, polyoxyethylene ether of glycerin ester, polyoxyethylene ether of Sorubita N'esuteru and sorbitol esters, Polyethylene ethylene ether can be shown as an ether ester type nonionic surfactant.
  • Ether ester type nonionic surfactants include polyoxyethylene glycerin fatty acid ester, castor oil, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene fatty acid alkanolamide sulfate, etc. .
  • a particularly preferred ether ester type nonionic surfactant is polyoxyethylene sorbitan fatty acid ester.
  • commercially available non-ionic surfactants that can be used in the composition of the present invention will be exemplified.
  • NIKKOL BL-9EX Polyoxyethylene (9) Lauryl Ether
  • NP-40 Polyoxyethylene (9) Octylphenyl Ether
  • TWEEN 20 Polyoxyethylene (20) Sorb i tan Monolaurate
  • TWEEN 80 Polyoxyethylene (20) Sorbitan Monooleate
  • TWEEN 40 Polyoxyethylene (20) Sorbitan Monopalmitate
  • TWEEN 60 Polyoxyethylene (20) Sorbitan Monostearate
  • TWEEN 85 Polyoxyethylene (20) Sorbitan Trioleate
  • anionic surfactant examples include surfactants such as a sulfonate, a carbonate, a sulfate, and a phosphate.
  • the following detergents can also be used in the composition of the present invention. These detergents can also use salts other than sodium salts.
  • the concentration of the denaturing agent in the reaction solution can be appropriately adjusted depending on the base sequence constituting the primer to be agglutinated and the temperature of the complementary strand synthesis reaction. Generally, 0.01% W / V to 10% W / V, for example, 0.5% W / V to 5% W / V, more specifically, 1% W / V to 3% W / V V can be added.
  • the buffer composition of the present invention may contain additional components used for complementary strand synthesis of nucleic acids. Examples of additional components are given below.
  • dXTP dATP, dTTP, dCTP, and dGTP
  • An enzyme protecting agent is a compound added for the purpose of protecting an enzyme that catalyzes complementary strand synthesis.
  • an inactive protein such as serum albumin or a surfactant such as DMS0 has a protective effect on the enzyme.
  • the buffer composition of the present invention may contain a substance necessary for analyzing a product of complementary strand synthesis.
  • the intercalator is an important indicator in the analysis of reaction products.
  • a compound that binds to a double-stranded nucleic acid to generate a signal is called an intercalator.
  • interlators are ethidium bromide (brominated by chemical bromide) and as an available product, cyber green (manufactured by Molecular Probe).
  • a primer for complementary strand synthesis can be added to the buffer composition of the present invention in advance.
  • the buffer composition of the present invention is useful for nucleic acid complementary strand synthesis.
  • Primers for complementary strand synthesis are appropriately designed according to the base sequence of the nucleic acid to be type III.
  • the nucleic acid can be distributed in the buffer composition of the present invention while containing a primer for complementary strand synthesis.
  • a primer having a base sequence specified by the user can be synthesized and incorporated into the buffer composition of the present invention.
  • the buffer composition of the present invention can be supplemented with a nucleic acid synthetase that catalyzes phase capture synthesis.
  • a nucleic acid synthetase that catalyzes phase capture synthesis.
  • complementary strand synthesis can be started only by adding a type III nucleic acid.
  • a desired complementary strand synthesis reaction can be started by adding a primer and a nucleic acid to be a type II. Any of these additional components can be added at the concentrations required for the final reaction.
  • the buffer composition of the present invention can be supplied in any dosage form.
  • It can be liquid, powder, or tablet.
  • Liquid preparations include those adjusted to the final concentration and those concentrated.
  • a liquid concentrated composition containing each composition at a 10-fold concentration is diluted 10-fold before use.
  • the solid components contained in the buffer composition of the present invention can be mixed and supplied in the form of a powder or a tablet.
  • the user can obtain a buffer composition adjusted to the final concentration by dissolving the powder or tablet with the specified volume of solvent.
  • Tris-HCl is used as a buffer
  • Tris-HC1 can be specified as a solvent for dissolution.
  • the buffer composition of the present invention can be used as a reaction solution for a complementary strand synthesis reaction involving primer annealing.
  • the buffer composition of the present invention is particularly useful when an oligonucleotide having a small number of constituent bases is used as a primer.
  • annealing of the primer is performed at a temperature about 5 ° C lower than Tm.
  • oligo nucleotides with a small number of constituent bases have low Tm.
  • nonspecific annealing and the higher-order structure of the oligonucleotide prevent phase capture synthesis from proceeding. Or, even if the conditions that allow complementary strand synthesis are given, it is difficult to synthesize the target synthetic product specifically because non-specific synthetic products are conspicuous. These obstacles can be eliminated by using the buffer composition of the present invention.
  • the GP method As a method for analyzing a nucleic acid requiring a complementary strand synthesis reaction using an oligonucleotide having a small number of constituent bases as a primer, the GP method can be shown. As shown in FIG. 1, in the GP method, an aggregate of phase capture chains synthesized using a plurality of regions of a test nucleic acid as ⁇ is analyzed. One method for synthesizing a plurality of regions is to anneal a primer having a small number of constituent bases to a plurality of locations. If the number of bases constituting the primer is small, the possibility that a nucleotide sequence complementary to the primer is present in the test nucleic acid is increased. As a result, a single primer can act as a primer in multiple regions. In implementing the GP method, for example, a plurality of regions to be subjected to phase capture synthesis are selected based on the following concept.
  • the plurality of regions include regions in which it is expected that there is a difference in base sequence between nucleic acids to be compared.
  • the difference in base sequence may not be included in all of the plurality of regions. For example, suppose that three nucleic acids X, Y, and ⁇ are synthesized for three regions a-c. 'Indicates that the base sequence of each region contains a difference.
  • the nucleic acid Y has b ′ having a base sequence different from that of other nucleic acids, and the nucleic acid Z has By combining these areas, the three parties can be identified. '
  • the plurality of regions specifically extract nucleotide sequence information as diverse as possible from a test nucleic acid.
  • nucleotide sequence information as diverse as possible from a test nucleic acid.
  • the base sequences differ from each other in as many regions as possible.
  • nucleotide sequence information of the genome has already been clarified.
  • a region having a different base sequence between the species to be compared and the other species can be selected in advance. Further, even when the base sequence information of the genome is insufficient, it is possible to select a region having a different base sequence based on the genome or gene information that has already been clarified.
  • the plurality of regions are synthesized by a method having reproducibility.
  • the reproducibility of complementary strand synthesis means that the same region is used as type II of complementary strand synthesis.
  • the length of the synthesized product can be changed.
  • the reproducible complementary strand synthesis reaction in the present invention can be said to mean that a certain primer specifically initiates complementary strand synthesis. Therefore, the complementary strand synthesis reaction of the present invention is distinguished from a complementary strand synthesis reaction using a random primer.
  • each nucleic acid should be synthesized under the same conditions. Therefore, when a plurality of regions are synthesized by a complementary strand synthesis reaction using a primer and a DNA polymerase, it is desired that the specificity of hybridization of the primer be maintained.
  • the method for synthesizing a nucleic acid is not particularly limited as long as it is a type-dependent synthesis method.
  • the plurality of regions can be synthesized using a sense strand or an antisense strand as a type III. All of the plurality of regions may be synthesized with the sense strand or the antisense strand as type III, or both may be synthesized as type III.
  • an extension product for the GP method As a typical method for obtaining an extension product, a phase capture synthesis reaction using a DNA polymerase can be used.
  • a primer capable of annealing to a plurality of regions of the test nucleic acid is used in order to synthesize a plurality of regions of a test nucleic acid.
  • an extension product synthesized by a complementary strand synthesis reaction from a primer capable of annealing to a plurality of regions of a test nucleic acid is called an extension product.
  • the extension product is an aggregate of polynucleotides synthesized using a plurality of regions of the test nucleic acid as type III.
  • the nucleotide sequences of the polynucleotide chains constituting the extension product are different from each other.
  • a plurality of polynucleotide chains containing the same base sequence are generated.
  • test nucleic acids having the same nucleotide sequence in different regions are designated as type III, an extension product containing the same nucleotide sequence may be generated.
  • the base sequence of the extension product since it is desirable to obtain a versatile waveform pattern of the dissociation curve, it is desirable to design the base sequence of the extension product so as to include as much difference as possible.
  • a primer that anneals to a plurality of regions of a test nucleic acid and enables complementary strand synthesis of the plurality of regions is referred to as a waveform generation primer in the GP method.
  • the waveform generating primer of the GP method is used for complementary strand synthesis of a plurality of regions of a test nucleic acid, and gives a mixture of extension products having different base sequences as a result of phase capture chain synthesis.
  • the mixture of different extension products in the GP method preferably refers to a mixture of polynucleotides that give a plurality of Tms when a dissociation curve is analyzed.
  • the primer for waveform generation in the GP method can also be defined as follows. That is, the primer for generating a waveform in the GP method is an oligonucleotide containing a nucleotide sequence complementary to a specific nucleotide sequence region appearing at a plurality of positions on a test nucleic acid, and the nucleic acid to be detected under the same conditions. In the above plurality of regions, the mirror-dependent DNA polymerase can initiate complementary strand synthesis. At least a part of the nucleotide sequence of the waveform generating primer comprises a nucleotide sequence complementary to the specific region. The base sequence complementary to the specific region can be arranged in any region of the oligonucleotide.
  • a primer capable of annealing to a plurality of regions of a test nucleic acid for example, the following method can be shown.
  • primers By designing primers based on the following concept, primers can be synthesized more easily than simply mixing primers necessary for the synthesis of complementary strands in multiple regions.
  • a single primer can anneal to a plurality of sites of a test nucleic acid by selecting an appropriate base sequence.
  • an oligonucleotide of 20 to 50 bases is used as a primer. Oligonucleotides of this length are very unlikely to anneal to multiple sites under stringent conditions.
  • oligonucleotides with a smaller number of bases increases the possibility of annealing at multiple positions.
  • region oligonucleotide consisting 3-8 bases can Aniru is computationally will Rukoto exist at a probability of 1/64 (4 3) to 1/65536 (4 8).
  • Rukoto exist at a probability of 1/64 (4 3) to 1/65536 (4 8).
  • a short primer enables the complementary strand synthesis of multiple regions even when used alone.
  • the design of one type of primer that can be annealed to multiple regions involves searching for a short base sequence consisting of 3 to 10 or more bases that appear frequently in the target genome based on the base sequence information of the target genome. This is possible by using the top candidates in descending order of appearance frequency.
  • the genome region for which the frequency of appearance is counted may include all nucleotide sequence information or may be targeted to a specific region of interest.
  • Two or more primers that anneal to multiple regions Two or more primers that anneal to multiple regions:
  • the base sequence of the nucleic acid to be type III is known, it is easy to design a plurality of primers that anneal to each site in order to synthesize a plurality of regions.
  • phase capture chain synthesis may be necessary for more regions.
  • the number of regions to be synthesized increases, the number of required primers also increases.
  • the region to be synthesized is selected so that the base sequences between the primers are as common as possible.
  • two regions for primer annealing are selected.
  • These nucleotide sequences are composed of two types of acid-fast bacilli A ( ⁇ co 3 ⁇ 4 cterium's) and B ⁇ Mycobacterium kansasii). Even though the differences are in different positions on the genome, the base sequences have high similarity to each other (matching bases are shown in capital letters).
  • Mycobacterium A AGcTcGTAAa (SEQ ID NO: 1)
  • Mycobacteria A AGtTcGTAAt (SS column number: 2)
  • Mycobacterium B AGgTtGTAAa (SEQ ID NO: 3)
  • Mycobacterium B AGtTcGTAAa (SEQ ID NO: 4)
  • the bases at the third, fifth, and tenth bases are different, and the other bases are identical. If these different bases are represented by an ambiguity code, it will be agBtYgtaaW (SEQ ID NO: 5).
  • an ambiguous code is a symbol for assigning a plurality of bases to a certain position.
  • the oligonucleotide represented by the ambiguous code means a mixture containing a combination of all bases represented by the ambiguous code at the position of the ambiguous code.
  • the ambiguous codes used here respectively correspond to the following bases.
  • a primer having a base sequence represented by an ambiguous code is referred to as an ambiguous primer.
  • primers are required for each region in order to synthesize complementary chains of the four regions.
  • the complementary strand synthesis of four regions became possible by one kind of polysemy primer.
  • Ambiguous primers are a collection of oligonucleotides having different base sequences. However, in operation, it is synthesized by the same operation as a single oligonucleotide, and can be used as a single oligonucleotide in complementary strand synthesis.
  • nucleotide sequence candidates with a high frequency of appearance for each of a plurality of regions of interest by selecting a nucleotide sequence with a higher degree of commonality from among the nucleotide sequences between the upper candidates, a plurality of completely independent nucleotide sequences can be selected. This makes it possible to design a single type of ambiguous primer that can obtain the same information at the same time.
  • degenerate primers In general, when a part of the base sequence of a type III nucleic acid is unknown, a degenerate primer is often used.
  • the base sequence of the degenerate primer is represented in the same manner as the polysequence primer in the present invention.
  • ambiguous primers are intended for the synthesis of multiple regions having different base sequences.
  • degenerate primers used for PCR cloning and the like usually aim at synthesizing one type of nucleic acid for one type of ⁇ . For crawling (ie, gene isolation), primers that produce a variety of synthetic products are undesirable.
  • the purpose of the design is completely different between the ambiguous primer and the degenerate primer.
  • degenerate primers can be used as waveform generating primers in the GP method.
  • the design method depends on the test nucleic acid. Therefore, application may be difficult depending on the nucleotide sequences of the test nucleic acid and the nucleic acid to be compared.
  • the method described below is a method in which a plurality of primers can be designed relatively easily without being affected by the base sequences of the test nucleic acid and the nucleic acid to be compared.
  • the ambiguous primer was arranged with an ambiguous code according to the base sequence of the nucleic acid to be ⁇ -shaped.
  • a polysemy code is placed on the 5 'side of the primer to obtain a primer assembly for waveform generation that can anneal to multiple regions of the test nucleic acid. Can also be. That is, in the GP method, a primer assembly for generating a waveform containing the following specific primers and ambiguous primers can be used.
  • Specific primer Includes a complementary base sequence in the target region of type I nucleic acid
  • Ambiguous primer at least one ambiguous primer containing the following specific region and ambiguous region
  • Specific region Contains the 3 'end of the oligonucleotide and is composed of a base sequence complementary to the target region
  • Ambiguous region Includes a base sequence that is located on the 5 'side of the specific region and in which a base that constitutes a base sequence complementary to the target region is replaced with a base other than the base
  • the specific primer in the GP method comprises an oligonucleotide containing a base sequence complementary to a target region of type I nucleic acid or a derivative thereof.
  • the derivative includes an oligonucleotide having an additional base sequence or a modified oligonucleotide.
  • Oligonucleotides can be modified with fluorescent materials, radioactive materials, or binding ligands.
  • the target region in the GP method refers to a region where a primer for phase capture strand synthesis should be annealed to a type I nucleic acid.
  • the target region is a region located on the 3 ′ side of the region to be synthesized for the type III nucleic acid.
  • the type ⁇ nucleic acid to be synthesized is a region selected as a region in which a waveform pattern specific to a test nucleic acid can be expected in the nucleic acid identification method using the GP method. It is desirable. Such a region can be selected centering on a region containing a difference in base sequence from the nucleic acid to be compared.
  • a highly homologous nucleotide sequence found at a different position can be selected as a target region.
  • a primer that can anneal to a region having low homology to the target region can be provided by using a set of primers for waveform generation based on the GP method.
  • a waveform is generated that enables complementary strand synthesis of multiple regions of type I nucleic acid.
  • a primer assembly can be obtained.
  • a region that does not include the polymorphic code on the 3 ′ side is a specific region
  • a region where the polymorphic code on the 5 ′ side is arranged is a polymorphic region.
  • the specific region is composed of the same nucleotide sequence as the nucleotide sequence constituting the region including the 3 'end of the specific primer. That is, the base sequence including the 3 'end of the ambiguous primer and the specific primer is common.
  • the number of bases constituting the specific region is not limited. The length of the unique region can be set as appropriate according to the conditions of the ambiguous region.
  • the length of the specific region can be selected within the range of 20 to 90% of the total length of the ambiguous primer.
  • the polysemy code constituting the polysemy region can be, for example, a sequence of four nucleotide polysemy (N), or the polysemy code necessary as appropriate as described in the example of acid-fast bacterium according to the nucleotide sequence of the test nucleic acid. Can also be arranged. Further, as described below, the variation of the polymorphic region can be increased stepwise from the 3 'side to the 5' side of the primer.
  • the ambiguous primer designed in this manner anneals with high reproducibility to many regions of the test nucleic acid, and provides a complementary strand synthesis product of a plurality of regions.
  • the polysemy on the 3 'side which determines the reaction specificity, is reduced, but the specificity is relatively unaffected 5' the 5 C such structural features can give diversity to the base sequence of the primer at the side, can be a collection of stably primers capable and child Aniru for a variety of base sequences.
  • the individual oligonucleotides constituting the primer assembly are designed to have low 3 'diversity. As a result, it selectively anneals to a base sequence that is complementary to the base sequence of each oligonucleotide, and initiates complementary strand synthesis. In other words, complementary strand synthesis can be started specifically and stably for a wide range of base sequences.
  • the following structure is used as a polysemy primer in which the polysemy in the polysemy region is gradually changed. You can show the structure.
  • the base sequence of the ambiguous region consists of the following three regions, and an aggregate of polysemy primers containing all combinations of the substituted base sequences constituting each region is useful as a primer assembly for waveform formation in the GP method It is.
  • N region constitutes the 5 'end of the polymorphic region, and each base constituting the base sequence is selected from adenine, cytosine, guanine, and thymine instead of a base complementary to the base sequence of the target region. Is a base substituted with all three types of bases other than the base
  • each base constituting the base sequence is replaced with a base complementary to the base sequence of the target region, and is replaced with adenine, cytosine, guanine, and thymine Is a base substituted with any two types of bases other than the base selected from
  • each base constituting the base sequence is replaced with a base complementary to the base sequence of the target region, and is replaced with adenine, cytosine, guanine, and It is a base that is substituted with any one type of base other than the base selected from thymine
  • the polysemy region is designed so that the polysemy increases stepwise from the 5 ′ end to the 3 ′ end. That is, at the 5 'end, there is an N region composed of N, which is an Ambiguity Code indicating one of A (adenine), C (cytosine), G (guanine), and T (thymine). .
  • the N region can also be said to be a region in which a base complementary to the target region has been replaced with any of the other three bases.
  • 3 ambiguity regions are arranged in which the base complementary to the target region is replaced by two other types of bases. Further, 2 ambiguity regions (2ARs) in which bases that are complementary to the target region are replaced with one other type of base are placed on the 3 ′ side of the 3AR.
  • the two Ns on the terminal side constitute the N region
  • the four bases of V, H, D, and B constitute the 3AR c.
  • the two S's on the 3 and 2 sides constitute a 2AR.
  • polysemy region polysemy can be enhanced from the 3 side to the 5 'end. In other words, such a structure can increase specificity from the 5 'end to the 3' end.
  • Table 1 shows the correspondence between the ambiguous codes used in the GP method and the actual bases.
  • the length of the N region is preferably 2 to 4 bases.
  • the number of bases in the N region is 5 or more, there is a high possibility that primer interference such as a dimer or a loop easily occurs.
  • the 3AR sequence is more stable in the order of DHVB, HDVB, VHDB, and HVDB.
  • the higher the proportion of G or C on the third side the higher the stability.
  • the 2AR sequence also needs to be designed in consideration of the base sequence of the target region.
  • the length ratio of the N region, 3AR, and 2AR is preferably 1: 2: 1, but is not limited thereto.
  • the length of the ambiguous region of the ambiguous primer constituting the primer assembly is preferably 10% to 80% of the number of bases constituting the primer.
  • the total length of the specific region and the polymorphic region of the polymorphic primer constituting the primer assembly is preferably 10 to 30 bases.
  • a primer assembly for generating a waveform by the GP method including a polymorphic primer can be designed based on the base sequence of the region by setting at least one region as the target region.
  • a plurality of target regions can be selected.
  • the types of specific primers increase, and the types of polysequence primers increase accordingly.
  • the primer assembly for waveform generation can be used simultaneously or individually for complementary strand synthesis.
  • each of the waveform generation primer aggregates has 15. If a shape pattern can be obtained, it means that 50, 625 types (15 X 15 X 15 X 15) of waveform patterns can be obtained theoretically. This means that the method of identifying nucleic acids based on the GP method can identify more than 50,000 nucleic acids by using four sets of primers for generating waveforms that can generate five types of waveform patterns. Is shown.
  • a plurality of types of waveform-generating primer aggregates designed for a plurality of regions can be used as a mixture of aggregates or independently for each aggregate for complementary strand synthesis.
  • the relationship between the waveform pattern and the set of primers can be clearly understood.
  • the primer assembly A for waveform generation designed based on a certain region A and the primer assembly B for waveform generation designed based on another region B are separately mixed without phase capture synthesis. Is preferably used.
  • the primer can be annealed to the mirror nucleic acid
  • a substrate is provided for complementary strand synthesis.
  • the buffer composition of the present invention when the number of constituent bases of the primer is small and a low Tm has to be adopted, the use of the buffer composition of the present invention is effective.
  • the components necessary to enable annealing at a particularly low temperature are the following components d) and e). Therefore, according to the present invention, there is provided a method for hybridizing an oligonucleotide to a complementary strand at a low temperature in the presence of one or both of the following components d) and e).
  • Consists of 0.11-0.1 M sulfate, sulfite, phosphate, and carbonate At least one salt selected from the group
  • the low temperature refers to, for example, a temperature at least 10 ° C lower than the Tm of the oligonucleotide to be hybridized. More specifically, the low temperature in the method of the present invention is, for example, 10 to 30 ° C., preferably 15 to 25 ° C., or 20 to 30 ° C. compared to the Tm of the oligonucleotide to be annealed. Includes temperatures 25 ° C lower.
  • the Tm of the oligonucleotide can be calculated according to the Wallace method described above.
  • Oligonucleotides requiring such low temperatures usually have a length of 5-20 bases, for example 7-15 bases. Therefore, oligonucleotides having such a length are preferred as oligonucleotides to which the method of the present invention is applied.
  • the length of Tm and the length of the oligonucleotide should be calculated based on the length of the base sequence constituting the portion that hybridizes to type III.
  • Oligonucleotides used as primers often have an additional nucleotide sequence independent of anneal.
  • a promoter or a recognition sequence for a restriction enzyme is usually a nucleotide sequence unrelated to primer anneal. Such a nucleotide sequence unrelated to anneal is not included in the number of bases subjected to Tm anneal.
  • the components d) and e) used in the method of the present invention can be used based on the conditions described above. Further, the method of the present invention is useful in a complementary strand synthesis reaction using a short primer, such as the GP method. Further, the present invention provides a stabilizer for oligonucleotide hybridization, which contains any one or both of the above-mentioned components and e) as an active ingredient. The stabilizer of the present invention is particularly useful for stabilizing the hybridization of an oligonucleotide having a small number of constituent bases. The method of the present invention or the stabilizer of the present invention Can be used for the method. For example, the short Annealing of primers is a typical example to which the method of the present invention can be applied.
  • the method of the present invention or the stabilizer can be used.
  • the method of the present invention or the stabilizing agent can also be applied when using a short oligonucleotide as a probe in various hybridization assays.
  • the method of the present invention, or the d) salts or e) denaturant constituting the stabilizer may be used alone or as a mixture of a plurality of compounds. It is considered that each of the compounds is stabilizing the primer ayur or improving the specificity by a different mechanism. Therefore, the effect of the stabilizer is enhanced by adding the surfactant, the detergent, and the salts.
  • the present invention relates to the use of the above components d) salts and e) denaturing agent in a phase capture synthesis reaction using short primers under low temperature conditions.
  • the low temperature condition in the present invention refers to a condition that is at least 10 ° C. lower than the Tm of the oligonucleotide to be hybridized. More specifically, the low temperature in the method of the present invention is, for example, 10 to 30 ° C., preferably 15 to 25 ° C., or 2 to 10 ° C. compared to the Tm of the oligonucleotide to be annealed. 0 to 25 ° C lower temperatures are included.
  • the short oligonucleotide according to the present invention usually has a length of 5 to 20 bases, for example, 7 to 15 bases.
  • FIG. 2 shows a conceptual diagram of the ambiguous primer.
  • the polysequence primer of Fig. 2 has a polysequence region at the 5 'end, so that it can anneal not only to a specific base sequence but also to a similar sequence similar to this. Can be.
  • a primer with a unique region and a unique region of 1: 1 was used at 98 ° C / 2 seconds, 40 ° CZ20 seconds, and 72 ° C / 20 seconds.
  • the protocol was carried out for 50 cycles to perform a complementary strand synthesis reaction.
  • the dissociation curve waveform of the extension product obtained by complementary strand synthesis was analyzed, and the effect of the polymorphic primer structure on the results was compared.
  • Ambiguous primers were evaluated based on two indicators of specificity and stability.
  • “highly specific” means that it is difficult to anneal to a region other than the portion selected as the target region.
  • the high specificity of the ambiguous primer causes a decrease in the diversity of extension products. As a result, the diversity of the waveform is lost. That is, it can be said that a polysemy primer having a reduced specificity is a desirable polysequence primer in the present invention.
  • the ambiguous primer is an aggregate composed of oligonucleotides having different base sequences.
  • the individual oligonucleotides that make up the aggregate The tide should anneal to the base sequence complementary to each base sequence.
  • the length of the ambiguous region is too long, the amount of the oligonucleotide corresponding to the target base sequence will decrease. That is, the possibility that sufficient complementary chain synthesis does not occur increases.
  • the specificity or stability of the primer was improved, but the diversity of the obtained dissociation curve was reduced. It was considered that the increased specificity did not sufficiently promote the phase capture synthesis reaction in a region other than the region selected as the target region.
  • the total primer length is 10 to 16 mer, the diversity of the waveform increases. However, specificity and stability have declined.
  • the total primer length was 9 mer or less, no synthesis reaction occurred. From these results, it was concluded that the range of 10 to 30 mer can be used as the primer length of the ambiguous primer of the present invention, and that the preferred length is 16 to 24 mer.
  • the ratio of the polymorphic region to the total length of the primer was 0.55 to 0.88, the waveform diversification increased but the stability decreased. If the conditions at this time are expressed in terms of the number of bases, the polymorphic region is 14 to 25 mer, and the specific region is 4 to 7 mer. In addition, when the ratio of the ambiguous region to the total primer length was 0.12 to 0.33, the diversity of the waveform was lost. If the conditions at this time are indicated by the number of bases, the polymorphic region is 4 to 7 mer, and the specific region is 14 to 25 mer.
  • the ratio of the polymorphic region to the total primer length is 0.89 or more, It was a random primer and did not undergo normal synthesis. If Shimese the conditions in the salt base, for example, ambiguous region 2 5 mer or more, and c also unique region is 3 mer or less, at a rate of ambiguous regions occupying the primer entire length to zero. 1 1 below the usual A synthesis reaction similar to the PCR primer was observed. In other words, almost only one type of complementary chain synthesis product was found. If the conditions at this time are represented by the number of bases, for example, the polymorphic region is 3 mer or less, and the unique region is 25 mer or more.
  • the ratio of the polymorphic region to the entire length of the primer is 0.333 to 0.55.
  • the specific region has a base sequence complementary to a target region selected from the test nucleic acid.
  • the length of the specific region is preferably 8 to 13 mer when the total length of the ambiguous primer is 16 to 24 mer.
  • the percentage of G C, which is occupied by G (guanine) or C (cytosine) in 5 bases including the 3 ′ end is 50% or more.
  • the stability of the terminal must be 15.5 to 19.5 kcal / mol or more.
  • annealing is possible even if there is a mismatch of 50% or less at the bases other than the 3 terminal 3 bases. At this time, if the stability of the mismatched portion is 12.0 Kcal / mol or less, the decrease in the synthesis efficiency can be ignored.
  • the ambiguous region is a region arranged to increase primers that can be annealed to a region other than the target region selected from the test nucleic acid.
  • the polysemy region has a base sequence in which a base complementary to the target region is replaced with another base.
  • the type of the base to be substituted is one to three arbitrary bases.
  • an ambiguity code shown in Table 1 is used to express the substitution of a base.
  • the structure of the ambiguous primer can be represented by a base sequence as shown in FIG. 3, for example.
  • the polysequence primer having the nucleotide sequence shown in FIG. 3 has a structure in which the polysemy in the polysemy region gradually decreases from 5 ′ to 3 ′. Length of ambiguous area
  • the primer is preferably 83mer when the total primer length is 16 to 24mer.
  • the above-mentioned aggregate of ambiguous primers including an ambiguous region is actually an aggregate of oligonucleotides having different base sequences. Therefore, the inclusion of one 2Ambiguity Code means that two kinds of oligonucleotides having different nucleotide sequences are actually contained. Therefore, the effective rate of annealing with type I nucleic acid is halved.
  • the base sequence SATT containing 2 Ambiguity Code: S (indicating C or G) means that it actually contains an oligonucleotide consisting of two types of base sequences, CATT and GATT. In this case, the amount of each effective primer is halved. Similarly, one 3 Ambiguity Code or 4 Ambiguity Code results in an effective primer amount of 1/3 or 14 respectively.
  • a sequence containing 3Ambiguity Code: B (indicating C, G or T) means that it contains an oligonucleotide consisting of three types of base sequences, CATT, GATT or TATT. I do.
  • the minimum primer amount is multiplied by the reciprocal of the effective primer amount, and this is divided by the number of bases in the polymorphic region and the mismatch rate of 4.
  • the number of bases in the ambiguous region is eight.
  • the minimum amount of primer is 1 ( ⁇ / ⁇ )
  • the method for calculating the amount of primer is an example, and is not limited to this method.
  • the Tm value refers to a temperature at which 50% of nucleic acids having complementary nucleotide sequences are in a base-paired state.
  • the Tm value of the specific region was calculated by the 2 (A + T) +4 (G + C) method, and the average of the polysemy region was calculated by the 2 (A + T) +4 (G + C) method. . These were combined to calculate the Tm value of the entire primer length.
  • the annealing temperature in the ordinary PCR method is about Tm minus 5 ° C.
  • the annealing temperature in the present invention is preferably set to a negative Tm value of about 20 ° C. in order to diversify the waveform pattern. Providing lower temperatures also makes it easier to anneal short primers. Experimentally, the divergence of the dissociation curve was significantly improved below 40 ° C. On the other hand, below 15 ° C, only random priming occurs, and almost no synthesis reaction is shown.
  • FIG. 1 is a diagram schematically illustrating a complementary strand synthesis method according to the present invention.
  • Figure 1 is a diagram schematically illustrating a complementary strand synthesis method according to the present invention.
  • (B) shows how a plurality of extension products form various mutual interference structures.
  • FIG. 2 is a diagram showing the structure of the polymorphic primer used in the present invention.
  • FIG. 3 is a diagram showing an example of the sequence of a polymorphic region of a polymorphic primer.
  • FIG. 4 shows the effect of the addition of a stabilizer on the waveform pattern in phase capture synthesis.
  • FIG. 4 (A) shows the waveform pattern without the stabilizer
  • FIG. 4 (B) shows the waveform pattern with the stabilizer added.
  • the vertical axis represents the fluorescence intensity differential value
  • the horizontal axis represents the temperature (° C).
  • FIG. 5 is a photograph showing the effect of the addition of a stabilizer in complementary strand synthesis on complementary strand synthesis.
  • FIG. 5 (A) shows the result of electrophoresis of the synthesized product when the stabilizer was not used
  • FIG. 5 (B) shows the result when the stabilizer was added.
  • the leftmost lanes of (A) and (B) are the 20 Obp ladder, and the two lanes on the right are the results of electrophoresis of sampnole (duplicate).
  • Fig. 6 schematically shows the positional relationship on the test nucleic acid of the ⁇ target region '' set for the design of the four types of waveform generation primer assemblies sPGBUP65, sPGBUPUPR, sPGBUPFX, and sPGBUPRX. .
  • stabilizers annealing stabilizers
  • Table 2 The effects of the annealing stabilizers (hereinafter referred to as “stabilizers”) shown in Table 2 will be described more specifically based on Examples.
  • stabilizers annealing stabilizers
  • Table 2 The present invention is not limited to the following embodiments, and various modifications can be made without departing from the gist of the present invention.
  • a primer, BAUP65 (SEQ ID NO: 6), that recognizes specific nucleotide sequences of bacterial DNA at multiple locations was created, and nucleic acids were identified based on the GP method.
  • the effect of the stabilizer was confirmed by comparing the waveform patterns with and without the stabilizer.
  • a reaction solution having a composition of “without stabilizer” and “with stabilizer” 50 ⁇ of each sample was added to perform a complementary strand synthesis reaction. The reaction was performed with a duplicate gate, and the waveforms of a total of four samples were observed. Using iCycler (manufactured by Biorad), a protocol of 98 ° CZ for 2 seconds, 25 ° C / 40 seconds, and 72 ° C / 10 seconds was performed for 50 cycles to synthesize a phase capture chain. After the reaction, the temperature of the temperature range of 75 to 95 ° C and the temperature step of 0.1 ° C were observed at each temperature for 8 seconds to draw the waveform of the dissociation curve of the synthesized product.
  • iCycler manufactured by Biorad
  • a buffer which enables specific annealing of a primer under low-temperature conditions. If the buffer of the present invention is used, a base-sequence-specific phase-trapping synthesis reaction can be expected even when a primer composed of a short base sequence of, for example, about 10 bases is used.
  • the use of short primers makes it possible to synthesize multiple regions of a test nucleic acid with a small number of primers.
  • the buffer of the present invention is useful for a nucleic acid analysis method that requires phase capture synthesis of a plurality of regions, such as the GP method.
  • the buffer of the present invention can be used in a phase capture synthesis reaction using a short primer. Low temperatures are required for annealing of short primers.
  • the buffer of the present invention has made it possible to efficiently perform annealing and complementary strand synthesis reaction under low temperature conditions.
  • Short primers are advantageous when specifically synthesizing multiple regions with a small number of primers. By mixing and using specific primers for each region, it is possible to synthesize multiple regions in one reaction system. However, synthesizing multiple primers causes an increase in cost. Also, a plurality of primers having a large number of constituent bases may have different Tm. In such a case, the stringency cannot be matched among a plurality of primers.
  • the effect provided by short primers can be expected.
  • the PCR method it may not always be possible to design primers of sufficient length. That is, if the base sequence information to be synthesized is insufficient, it is not possible to design a primer having a sufficient length.
  • the number of bases for a complementary strand synthesis reaction in the primer may be limited due to a base sequence incorporated for a purpose other than the phase capture synthesis reaction.
  • primers that incorporate a promoter or restriction enzyme recognition sequence are often used. In such a case, a small number of bases are required as primers for the phase capture synthesis reaction. If the buffer of the present invention is used, even with such primers, the PCR method can be performed efficiently.
  • the buffer of the present invention By using the buffer of the present invention to synthesize complementary strands in a plurality of regions with short primers, the stringency between primers can be easily matched. In addition, when the primer is short, it is expected that a plurality of regions may share a primer having the same nucleotide sequence. As described above, the significance of the present invention which enables specific complementary chain synthesis using short primers is significant.

Abstract

L'invention concerne une composition tampon qui contient au moins un sel choisi dans le groupe constitué par les sulfates, les sulfites, les phosphates et les carbonates, et un modificateur. Ce tampon permet l'appariement des amorces et la synthèse d'une chaîne complémentaire, même dans des conditions de basse température. Il permet la synthèse d'une chaîne complémentaire spécifique du site à partir d'une amorce de longueur réduite, p. ex. 10 mer. Ce tampon convient pour le procédé GP (genopattern).
PCT/JP2004/007162 2003-05-20 2004-05-19 Composition tampon WO2004104196A1 (fr)

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PCT/JP2003/006275 WO2003097828A1 (fr) 2002-05-21 2003-05-20 Procede d'identification d'acides nucleiques
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JP2003-389821 2003-11-19

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7547514B2 (en) 2004-07-28 2009-06-16 Canon U.S. Life Sciences, Inc. Methods for monitoring genomic DNA of organisms
US7604938B2 (en) 2005-02-18 2009-10-20 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
US9394572B2 (en) 2009-07-24 2016-07-19 Morinaga Milk Industry Co., Ltd. Method and kit for detection of microorganism

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JPH09238687A (ja) * 1996-03-08 1997-09-16 Shimadzu Corp 核酸合成法
JPH1080279A (ja) * 1996-09-09 1998-03-31 Shimadzu Corp 核酸合成法
JP2000342287A (ja) * 1999-05-03 2000-12-12 Qiagen Gmbh リボ核酸類からの核酸類の生成及び増幅
JP2001299356A (ja) * 2000-04-27 2001-10-30 Shimadzu Corp 核酸合成法
JP2001352982A (ja) * 2000-06-12 2001-12-25 Shimadzu Corp 核酸合成法
WO2003097828A1 (fr) * 2002-05-21 2003-11-27 Adgene Co., Ltd. Procede d'identification d'acides nucleiques

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Publication number Priority date Publication date Assignee Title
JPH09238687A (ja) * 1996-03-08 1997-09-16 Shimadzu Corp 核酸合成法
JPH1080279A (ja) * 1996-09-09 1998-03-31 Shimadzu Corp 核酸合成法
JP2000342287A (ja) * 1999-05-03 2000-12-12 Qiagen Gmbh リボ核酸類からの核酸類の生成及び増幅
JP2001299356A (ja) * 2000-04-27 2001-10-30 Shimadzu Corp 核酸合成法
JP2001352982A (ja) * 2000-06-12 2001-12-25 Shimadzu Corp 核酸合成法
WO2003097828A1 (fr) * 2002-05-21 2003-11-27 Adgene Co., Ltd. Procede d'identification d'acides nucleiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7547514B2 (en) 2004-07-28 2009-06-16 Canon U.S. Life Sciences, Inc. Methods for monitoring genomic DNA of organisms
US7604938B2 (en) 2005-02-18 2009-10-20 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
US8841093B2 (en) 2005-02-18 2014-09-23 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic DNA of organisms
US9394572B2 (en) 2009-07-24 2016-07-19 Morinaga Milk Industry Co., Ltd. Method and kit for detection of microorganism
US10329604B2 (en) 2009-07-24 2019-06-25 Morinaga Milk Industry Co., Ltd. Method and kit for detection of microorganism

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