JP5924648B2 - Nucleic acid amplification method via PCR method - Google Patents

Description

  The present invention relates to a nucleic acid amplification method via PCR. Specifically, the present invention relates to a nucleic acid amplification method useful in genetic engineering, molecular biology, and related industrial fields.

  Nucleotide sequence polymorphism analysis including microbiota analysis is an important technique for medical diagnosis and phylogenetic classification of living organisms. However, the nucleotide sequence having a polymorphism may contain a very low content of mutations compared to the wild type, and in the analysis of microflora in the environment, a large amount of DNA of a specific microorganism is present. , It may make it difficult to detect the DNA of other microorganisms.

  Conventionally, in polymorphism analysis of SNP (single nucleotide substitution), there are several reported examples as a technique for preferentially amplifying a low-content mutant type compared to the wild type. For example, full-COLD-PCR (CO-amplification at Lower Generation temperature Polymerization Chain Reaction) provides a hybridization step to form heteroduplexes between wild-type and mutant DNA in the temperature change cycle of PCR. The wild type homoduplex DNA is not denatured into single strands, but re-denaturation is performed at a temperature at which the wild type-mutant heteroduplex DNA denatures, giving priority to the mutant type compared to the wild type. A method of amplifying the signal (see Non-Patent Document 1). In addition, fast-COLD-PCR uses a difference in Tm value caused by the presence of a mutation in a mutation in which the Tm value decreases (G> T, etc.), and undergoes heat denaturation only when there is a mutation. There is a method of preferentially amplifying the mutant type by using a temperature (Tc) that does not undergo thermal denaturation as a denaturing temperature in PCR (see Non-Patent Document 2).

  In full-COLD-PCR, due to the characteristics of the principle, it is difficult to increase the ratio of the wild type to the mutant type to 1: 1 or more, it takes a very long time for amplification, and the efficiency of selective amplification is low. In addition, although fast-COLD-PCR can be performed in a short time, each method has advantages and disadvantages such as being unable to cope with a mutation with an increased Tm value such as T> G. -COLD-PCR was developed (see Non-Patent Document 3). In ice-COLD-PCR, an oligonucleotide (Reference sequence: RS) that is complementary to the wild type and does not serve as a wild-type, mutant template or primer is present in an excess amount of the reaction solution. By providing a hybridization step in the same manner as above, a heteroduplex of wild-type RS and mutant-RS is formed. At this time, since the Tm value of the mutant-RS having a mismatch is reduced as compared with the wild-type RS of the perfect match, a denaturation temperature at which the mutant-RS is preferentially denatured is set in the PCR. Thus, it is a method of preferentially amplifying the mutant type. This development has made it possible to selectively amplify mutants in a relatively short time.

Li J et. al. Nat Med. 14 (5), 579-84 (2008) Li J et. al. Clin Chem. 55 (4), 748-56 (2009) Coren A. M.M. et. al. Nucleic Acid Research, 39 (1), e2 (2011)

  However, even in ice-COLD-PCR described in Non-Patent Document 3, since it is necessary to synthesize an RS corresponding to the wild type, selective amplification of mutants in a large number of polymorphism analysis and a large amount of microorganisms in the microflora Less versatile for selective exclusion of nucleic acid fragments.

  An object of the present invention is to provide a nucleic acid amplification method via a PCR method that improves the amplification efficiency of a mutant nucleic acid or a nucleic acid derived from a microorganism having a small abundance ratio when performing polymorphism analysis or microbial community structure analysis. is there.

The present invention relates to the following [1] to [4].
(1) mixing a double-stranded RNA having homologous nucleotide sequence to the sample and the DNA containing two or more DNA (A) containing DNA (A), after hybridization with heat denatured After maintaining the temperature at which the hybridization between the DNA other than the DNA ( A ) and the RNA is dissociated and the hybridization between the DNA ( A ) and the RNA is maintained, the dissociated DNA is amplified. DNA amplification method characterized by these.
[2] A method for detecting a single nucleotide polymorphism, wherein a gene polymorphism in a sample is detected using the DNA amplification method according to [1].
[3] A method for analyzing a microflora, wherein the microorganism- derived DNA in a sample is detected using the DNA amplification method according to [1].
[4] A method for improving the amplification efficiency of a low-content DNA fragment contained in a sample, using the DNA amplification method according to [1].

  According to the nucleic acid amplification method of the present invention, when polymorphism analysis or microbial community structure analysis is performed, amplification by a large amount of wild-type nucleic acid or nucleic acid derived from microorganisms growing at a main ratio is suppressed, and mutant nucleic acid or presence of nucleic acid is present. It becomes possible to improve the amplification efficiency of the nucleic acid derived from microorganisms with a small ratio. This improves the polymorphism analysis technique or the microbial community structure analysis technique, such as an improvement in sensitivity in polymorphism analysis and an improvement in detection sensitivity of a small number of microorganisms in the microbial community.

FIG. 1 is a schematic diagram of the nucleic acid amplification method of the present invention. FIG. 2 is a diagram showing an electrophoretic image of a denaturant concentration gradient gel electrophoresis (DGGE) method. Lane 1 is S. cerevisiae genome DNA and S. cerevisiae. An equimolar mixture of pombe genome DNA was amplified by a normal PCR cycle. Lane 2 is obtained by amplifying a DNA mixture prepared in the same manner as Lane 1 by the COLD-PCR cycle. FIG. 3 is a diagram showing a migration image of the DGGE method as in FIG. Lanes 1-5 are S.E. cerevisiae and S. cerevisiae. Pombe genomic DNA was mixed at a weight ratio of 1: 1, 10: 1, 100: 1, 1000: 1, 10000: 1, and the resulting DNA mixture was amplified by a normal PCR cycle. Lanes 6 to 10 are obtained by amplifying a DNA mixture prepared in the same manner by a normal PCR cycle so as to have the same cycle number as that of COLD-PCR. Lanes 11 to 15 are obtained by amplifying a similarly prepared DNA mixture by the COLD-PCR cycle. FIG. 4 is a diagram showing a migration image of the DGGE method as in FIG. The environmental genome obtained from red wine fermentation moromi is used as template DNA. Lanes 1 and 2 are DNAs obtained from the samples immediately after the addition of yeast on the day of red wine fermentation, and lanes 3 and 4 are the day 8 of starting red wine fermentation. DNA obtained from these samples is used. Lanes 1 and 3 are amplified by a normal PCR cycle, and lanes 2 and 4 are amplified by a COLD-PCR cycle. FIG. 5 is a diagram showing a migration image by agarose gel electrophoresis. Lane 1 is a molecular weight marker. Lane 2 is S. cerevisiae genome DNA and S. cerevisiae. An equimolar mixture of pombe genome DNA was amplified by a normal PCR cycle. Lane 3 is obtained by amplifying a DNA mixture prepared in the same manner as Lane 2 by the COLD-PCR cycle. Lane 4 shows the PCR product treated with the restriction enzyme KpnI. Lane 5 shows the COLD-PCR product treated with the restriction enzyme KpnI.

  The nucleic acid amplification method of the present invention synthesizes double-stranded RNA having a base sequence homologous to a nucleic acid (also referred to as nucleic acid A) for which amplification is not desired (suppression of amplification), and uses the double-stranded RNA as a sample. Addition and COLD-PCR are carried out, so that amplification of the nucleic acid A is suppressed and other nucleic acids are amplified. In the present specification, “nucleic acid” means both “DNA” and “RNA”, but “DNA” is preferred.

  More specifically, it is homologous to nucleic acid A in a solution of a sample containing nucleic acid (nucleic acid A) having an arbitrary specific base sequence and nucleic acid (referred to as nucleic acid B) that can be amplified with the same primer as nucleic acid A. By mixing and hybridizing nucleic acid fragments (double-stranded RNA) having a proper base sequence, homoduplex derived from nucleic acid A and double-stranded RNA, and heterogeneous derived from nucleic acid B and double-stranded RNA Double strands are formed. Next, the denatured single-stranded nucleic acid is selected and amplified by thermal denaturation at a temperature at which the homoduplex binding is maintained and the heteroduplex is dissociated into single-strands. It becomes possible. That is, it becomes possible to amplify only the nucleic acid B selected by the binding property with the double-stranded RNA by PCR. For example, in microbiota analysis, the amplification of nucleic acid fragments derived from a specific microorganism is suppressed, and other microorganisms The derived nucleic acid fragment can be amplified with higher sensitivity.

  Table 1 below shows the results of comparing the principles of the existing COLD-PCR and the COLD-PCR of the present invention.

  From Table 1, it is easy to prepare double-stranded RNA by using double-stranded RNA as a nucleic acid fragment having a base sequence homologous to a nucleic acid having an arbitrary specific base sequence. Conceivable. In addition, the Tm value of a perfect match DNA: RNA hybrid is usually higher than that of a double-stranded DNA of the same base length, compared to the case where a DNA strand of a nucleic acid that is not desired to be amplified is selected. The DNA: RNA hybrid to be suppressed is hardly dissociated while most of the amplified double-stranded DNA is dissociated, and the denaturation temperature can be set easily and efficiently compared to the case of using DNA. Amplification is possible. Furthermore, oligonucleotide synthesis usually has a limit of about 150 nt to 200 nt, so it cannot handle amplification products beyond that, but it can be used for lengths of about 600 bp by using double-stranded RNA. The effect is suggested.

  Hereinafter, the nucleic acid amplification method of the present invention will be described with reference to FIG.

  The sample used in the present invention is not particularly limited as long as it contains two or more kinds of nucleic acid containing nucleic acid A (main double-stranded DNA in FIG. 1) and can contain a nucleic acid that can be amplified with the same primer as nucleic acid A. Absent. For example, DNA can be directly extracted from environmental samples, foods, feces, and the like that constitute a certain type of microbiota, using a glass bead method, an enzymatic method, or a commercially available reagent in a kit. Hereinafter, nucleic acids other than nucleic acid A are referred to as nucleic acid B (low-containing double-stranded DNA in FIG. 1), and nucleic acid B may be a polymorphism of nucleic acid A.

  The double-stranded RNA having a base sequence homologous to the nucleic acid A is not particularly limited as long as it hybridizes to the DNA strand of the nucleic acid A in a complementary manner.

  Double-stranded RNA can be synthesized by using an RNA chemical synthesis method or various DNA-dependent RNA polymerases. For example, when DNA-dependent RNA polymerase is used, a nucleic acid fragment excised from a gel such as a denaturing gradient gel is used as a template, and a promoter sequence recognized by the DNA-dependent RNA polymerase, for example, a primer added with a T7 promoter sequence is used. Thus, double-stranded RNA can be synthesized using the amplified fragment as a template.

  In the microbiota analysis, a universal primer having a ribosomal RNA gene, for example, Small-SubUnit rRNA or Large-SubUnit rRNA sequence was used by denaturing gradient gel electrophoresis (hereinafter referred to as “DGGE”) method. A technique is often used in which a PCR product is separated for each base sequence and a microorganism species is estimated from the sequence of each DNA fragment. Therefore, in the present invention, a band of a nucleic acid fragment to be suppressed at that time can be cut out, and a double-stranded RNA can be synthesized by the above technique.

  The length of the double-stranded RNA is preferably 25 nucleotide residues or more, more preferably 50 nucleotide residues or more, preferably 1000 nucleotide residues or less, from the viewpoint of time required for hybridization and sequence selectivity. More preferably, it is 600 nucleotide residues or less.

  In the present invention, the composition containing the sample and double-stranded RNA is subjected to an amplification reaction by the PCR method. Therefore, the composition for performing the PCR reaction, that is, the PCR reaction solution is not particularly limited as long as it contains various components such as primers, buffers, and magnesium ions in an appropriate concentration in addition to the above components. It can be prepared according to methods well known to those skilled in the art. The concentration ratio of the sample (nucleic acid) to the double-stranded RNA (nucleic acid / double-stranded RNA) is not particularly limited because it cannot be determined in general depending on the type of nucleic acid and the like. 1000 is exemplified. In addition, in order to improve the selective amplification with the double-stranded RNA, the PCR reaction solution to be subjected to the amplification reaction using the double-stranded RNA is pre-amplified only with the target nucleic acid before adding the double-stranded RNA. Can be used.

  For example, the primer is not particularly limited as long as it is an oligonucleotide that anneals to nucleic acid A and nucleic acid B. In the present invention, a region that is well preserved among microorganisms may be used from the viewpoint of use for analysis of microflora in samples such as environment, food, and feces. Moreover, since it is preferable that the DNA fragment used for DGGE analysis is rich in GC base content, a primer to which a GC clamp (GC-Clamp) is added may be used. The primer chain length (the chain length excluding the GC clamp when added) is preferably 15 nucleotide residues or more, more preferably 20 nucleotide residues or more, preferably 30 nucleotide residues or less, more Preferably it is 25 nucleotide residues or less. The primer may be a commercially available product or synthesized according to a known method.

  The concentration of primer added cannot be determined in general depending on the type of primer and the concentration of double-stranded RNA used. From the viewpoint of compensating for the effective primer concentration that decreases due to annealing with double-stranded RNA, the final concentration of the reaction solution is 0.2 μM to 0.4 μM is preferable, and 0.3 μM to 0.4 μM is more preferable.

  In addition, when magnesium ions are contained, the concentration of the reaction solution is such that the final concentration of the reaction solution is from the viewpoint of compensating for the effective magnesium ion concentration that is reduced by binding of excessive amounts of RNA and magnesium ions contained in the reaction solution. 1.0 mM to 3.0 mM are preferable, and 1.5 mM to 2.5 mM are more preferable. What is necessary is just to adjust the said conditions suitably according to the density | concentration of double stranded RNA to add.

  The obtained PCR reaction solution is amplified via the PCR method according to a known COLD-PCR method (for example, full-COLD-PCR, ice-COLD-PCR). Specifically, the reaction solution is first subjected to heat denaturation I and then hybridized.

  In heat denaturation I, double-stranded DNA or double-stranded RNA in a PCR reaction solution is dissociated. The conditions for the heat denaturation incubation are not particularly limited, but examples of the temperature conditions include 94 ° C to 98 ° C, and 96 ° C to 98 ° C is more preferable. Moreover, as reaction time, 5 to 30 second is illustrated, and 10 to 15 second is more suitable. The conditions may be adjusted as appropriate according to the sample used and the amount of double-stranded RNA. In addition, from the viewpoint of sufficiently dissociating double-stranded DNA or double-stranded RNA in the PCR reaction solution before heat denaturation I, as heat denaturation (preheating), for example, at 94 ° C. to 96 ° C. for 1 minute. Incubation can be performed for ~ 3 minutes.

  Hybridization conditions are not particularly limited, but examples of temperature conditions include 70 ° C. to 80 ° C., and 70 ° C. to 75 ° C. are more preferable. Moreover, as reaction time, 30 seconds-10 minutes are illustrated, and 5 minutes-10 minutes are more suitable. The conditions may be adjusted as appropriate according to the sample used and the amount of double-stranded RNA.

  The reaction solution after hybridization contains a homoduplex derived from nucleic acid A and double-stranded RNA, and a heteroduplex derived from nucleic acid B and double-stranded RNA. Next, the reaction solution is subjected to heat denaturation II.

  In heat denaturation II, incubation is performed within a temperature range in which the hybridization between the nucleic acid B and the double-stranded RNA is dissociated and the hybridization between the nucleic acid A and the double-stranded RNA can be maintained. That is, homoduplexes derived from nucleic acid A and double-stranded RNA are not dissociated by incubation within the above temperature range, and only heteroduplexes derived from nucleic acid B and double-stranded RNA become single-stranded. Dissociated.

  In the present invention, from the viewpoint of efficiency of examination of PCR conditions, the Tm value of the DNA-DNA duplex and the Tm value of the DNA-RNA hybrid duplex for the nucleotide sequence of nucleic acid A that is to be selectively inhibited from amplification. Is preferably measured or calculated in advance. If the homology between the nucleic acid A and the other nucleic acid sequence (nucleic acid B) is low, the Tm value calculated by theoretical calculation from the salt concentration, base sequence, etc. may be used. In the present specification, the Tm value is the melting temperature of the oligonucleotide, and can be measured using Real-Time PCR or the like using a fluorescent dye such as SYBR GREEN I. Further, the Tm value can be calculated from the base sequence by using the closest base pair method or the GC% method.

  Examples of the incubation conditions in the heat denaturation II include 79 ° C. to 92 ° C. as the temperature conditions, 80 ° C. to 90 ° C. are more preferable, and 83 ° C. to 87 ° C. are more preferable. Moreover, as reaction time, 3 to 30 second is illustrated, 5 to 20 second is more preferable, and 10 to 15 second is further more preferable. The temperature is preferably about 10 to 15 ° C. higher than the hybridization condition. In the present invention, the heat denaturation II is also referred to as low temperature heat denaturation because of the difference in incubation temperature between the heat denaturation II and the heat denaturation I.

  Thus, the reaction solution after heat denaturation at low temperature includes single-stranded and double-stranded RNAs of homoduplex derived from nucleic acid A and double-stranded RNA, and nucleic acid B generated by dissociation of heteroduplex. Of single strands. Therefore, the nucleic acid B can be amplified by subjecting the reaction solution to annealing and extension reaction.

  The conditions for the annealing and extension reaction are not particularly limited, but when it is desired to simultaneously amplify those that may be mismatched as in the microbiota analysis, for example, the annealing temperature condition is 48 ° C. to 60 ° C. It is illustrated and 48 to 50 degreeC is more suitable. Further, examples of the annealing reaction time include 20 seconds to 30 seconds, and about 30 seconds are more preferable. The temperature condition in the extension reaction is preferably 68 to 72 ° C. Moreover, as reaction time in extension reaction, 30 second-1 minute / 1kb is illustrated, and when amplification object is 500 bp or less, about 30 second is more suitable. The conditions may be adjusted as appropriate according to the sample used and the amount of double-stranded RNA.

  Thus, one cycle of PCR reaction can be performed, and amplification of nucleic acid A can be suppressed and amplification of nucleic acid B can be improved. The obtained PCR reaction solution can be subjected to the heat denaturation I and the PCR reaction can be performed again. PCR may be performed at a temperature cycle of 30 to 40 cycles, for example.

  The method for quantifying and analyzing the obtained nucleic acid B is not particularly limited, and can be performed according to a known method. For example, a DNA fragment amplified by the COLD-PCR method can be further confirmed by a denaturing gradient gel electrophoresis (DGGE) method after further amplification by a normal PCR using a primer to which GC-Clamp has been added. .

  Thus, it becomes possible to selectively amplify low-containing double-stranded DNA (nucleic acid B) other than the main double-stranded DNA (nucleic acid A) contained in the sample. Therefore, for example, when an environmental sample is used, it is possible to grasp a small number of microbial groups in the microbial community structure, and thus it is possible to analyze the microbial flora.

  In the present invention, since the nucleic acid B can be selectively amplified as described above, the present invention also provides a method for improving the amplification efficiency of the nucleic acid B.

  In addition, when the nucleic acid B is a polymorphism of the nucleic acid A, the present invention provides a method for detecting the polymorphism.

  The polymorphism detection method of the present invention is not particularly limited as long as the PCR method is performed using a sample containing the polymorphism. For example, when a sample having a nucleic acid derived from a genome is used, gene analysis can be performed by analyzing the polymorphism of the amplified nucleic acid B.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following Example.

Example 1
The following experiment was performed using Saccharomyces cerevisiae (1003) and Schizosaccharomyces pombe (5315) as the model experimental system. Specifically, S.M. cerevisiae and S. cerevisiae. Each pombe was cultured in a YPD medium, and cells were obtained by centrifugation when the amount of cells exceeded 2 × 10 ⁸ cells / mL. Genome DNA was extracted from the obtained bacterial cells using Gen Toru-kun (registered trademark) (for yeast) High recovery (Takara Bio), and the purity and concentration were measured by NanoDrop 1000 (Thermo Fisher Scientific). The obtained genome DNA was mixed in an equal amount by weight ratio, and the concentration was adjusted so that the total content of DNA in the mixed solution was 10 ng / μL to obtain a template DNA sample.

  PCR uses KOD-plus Neo (TOYOBO), and thermal cycler uses GeneAmp (registered trademark) PCR System (Applied Biosystems) with the reaction solution composition shown in Table 2 (details of primers are shown in Table 3). went. In the temperature cycle, the first denaturation step (preheating) is performed at 96 ° C. for 3 minutes, and then the one-cycle denaturation step is 96 ° C. for 15 seconds, the annealing step is 50 ° C. for 30 seconds, and the chain extension step is 68 ° C. 40 cycles of 30 seconds were performed. Hereinafter, the temperature cycle is also referred to as “normal PCR cycle”. As a result, an amplification product of about 245 to 260 bases was obtained.

  The amplified product was subjected to DGGE method (D-code system, Bio-Rad) with a denaturant concentration gradient of 30% to 70% (denaturant concentration of 100% includes 7M urea and 40% formamide) at 100 V, 60 ° C., 16 hr. Electrophoresis, S. The cerevisiae band was cut out using a sterile Pasteur pipette. The obtained band was used as a template DNA, 40 PCR cycles were carried out with the same reaction solution composition as in Table 2 using a primer to which the T7 promoter sequence shown in Table 4 was added, and the resulting amplification product was converted into Nucleospin (registered trademark) Extract. Purified by II (MACHEREY-NAGEL). Next, using this as a template, double-stranded RNA was synthesized using in vitro transcription T7 Kit (for siRNA Synthesis, Takara Bio).

The synthesized double-stranded RNA is recovered as a pellet by ethanol precipitation, and after drying, DNase is removed in DNase-free DNase I (40 mM Tris-HCl, 0.66 mM MnCl ₂ ) buffer to remove the template DNA fragment. I treatment was performed at 37 ° C. for 2 hours. Subsequently, the double-stranded RNA was purified by NucleoSpin (registered trademark) RNA Clean-up Kit (MACHEREY-NAGEL), and 0.5% TBE (44.5 mM Tris, 44.5 mM boric acid (44.5 mM Tris) on a 10% acrylamide gel. Electrophoresis was performed at 100 V for 90 minutes using 1 mM EDTA (pH 8.0)), stained with GelRed (Biotium) for 30 minutes, and then the band was confirmed with a UV transilluminator. The purity, size, and concentration of RNA were confirmed by comparison with siRNA Ladder Marker (Takara Bio).

  Next, COLD-PCR was performed. First, a DNA template for COLD-PCR was prepared. Specifically, the previously prepared S.P. cerevisiae and S. cerevisiae. Using a pombe genome DNA mixed solution (concentration: 10 ng / μL) as a template, after performing 40 normal PCR cycles with primers having no GC-Clamp sequence shown in Table 5 and the same reaction solution composition as in Table 2, Was purified by Nucleospin (R) Extract II (MACHEREY-NAGEL).

The resulting amplification product was adjusted to a concentration of 1 ng / mu L, was PCR (COLD-PCR) in a reaction solution composition of Table 6 by using the primer set described in Table 5. In the temperature cycle, the first denaturation step (preheating) was performed at 96 ° C. for 3 minutes, then one cycle of the denaturation step (denaturation I step) at 98 ° C. for 10 seconds, the hybridization step at 75 ° C. for 10 minutes, Forty cycles of low temperature denaturation step (denaturation II step) at 86 ° C. for 15 seconds, annealing step at 50 ° C. for 30 seconds, and chain extension step at 68 ° C. for 30 seconds were performed (hereinafter, this temperature cycle is referred to as “ COLD-PCR cycle).

The resulting amplification product for analysis at the DGGE method, it is necessary to add a GC-Clamp to PCR amplified fragment after purification by re Nucleospin (TM) Extract II (MACHEREY-NAGEL) , 1ng / μ L After adjusting the concentration, 40 PCR cycles were usually performed with the reaction solution composition shown in Table 2 using the primers shown in Table 3 to obtain an amplification product (also referred to as COLD-PCR product).

On the other hand, as a contrast, S.I. cerevisiae and S. cerevisiae. genome DNA mixed solution of pombe (concentration: 10ng / μ L) as the template, using primers shown in Table 3 by performing 40 cycles of normal PCR cycle reaction solution composition shown in Table 2, the amplification products (both normal PCR products Say).

  Each obtained amplification product was electrophoresed on a denaturant gradient gel of 30% to 70% by the DGGE method. The results are shown in FIG. It is strongly detected from the amplification product of normal PCR. It was confirmed that the cerevisiae band (lane 1) was detected very weakly in the amplified product (lane 2) by COLD-PCR in which amplification was selectively suppressed using double-stranded RNA.

Example 2
S. cerevisiae and S. cerevisiae. Using pombe genome DNA, it was examined whether selective amplification can be seen no matter how much the initial abundance is different.

  Specifically, the genomic DNA extracted in Example 1 was S.p. cerevisiae: S. Mix so that the pombe is 1: 1, 10: 1, 100: 1, 1000: 1, 10000: 1, respectively, and adjust the concentration so that the total DNA content in the DNA mixed solution is 10 ng / μL. It was adjusted.

  In the same manner as in Example 1, normal PCR products and COLD-PCR products using primers with GC-Clamp were obtained.

  In addition to the above, the following examination was performed in order to examine the influence of the number of PCR cycles. Specifically, using the same genomic DNA mixed solution as described above as a template, 40 normal PCR cycles were performed with the reaction solution composition shown in Table 2 using primers without GC-Clamp (Table 5). Thereafter, 1 ng of the amplification product purified by Nucleospin (registered trademark) Extract II (MACHEREY-NAGEL) was used, and 40 normal PCR cycles were performed again using primers without GC-Clamp (Table 5) as described above. Next, 1 ng of purified by Nucleospin (registered trademark) Extract II (MACHEREY-NAGEL) was used, and further, 40 PCR cycles were performed using a primer with GC-Clamp (Table 3). Prepared (normal PCR product-2).

  Each obtained amplification product was electrophoresed on a denaturant gradient gel of 30% to 70% by the DGGE method. The results are shown in FIG. Lanes 1 to 5 are amplification products (normal PCR products) when PCR is performed directly from the genome DNA using primers with GC-Clamp, and lanes 6 to 10 are the same number of cycles as normal COLD-PCR. Amplification products (ordinary PCR products-2), lanes 11 to 15 are amplification products (COLD-PCR products) when COLD-PCR using double-stranded RNA is performed. In COLD-PCR, S. amplification of DNA fragments derived from S. cerevisiae is suppressed; S. cerevisiae, which exists only about 1 / 10,000. It was confirmed that the DNA fragment derived from pombe was amplified.

Example 3
In the wine fermentation process, since wine yeast is added in the initial stage, it contains a large number of yeast cells. Therefore, when cells are collected from the mash of the wine fermentation process, DNA is extracted and analyzed by the PCR-DGGE method. cerevisiae is detected as the main band. In such a state, it is not easy to detect an amplified fragment derived from a microorganism that is present only in a small number. Therefore, an S.C. Attempts were made to detect bands derived from microorganisms other than cerevisiae.

  On the first day after addition of red wine fermenting yeast and on the 8th day after yeast addition, glass beads were sampled from the mash, and from the precipitate fraction obtained by 10,000 × g, using nuclei lysis (promega) and protein prescription solution (promega). DNA was extracted by the method. The concentration of the extracted DNA was measured by NanoDrop 1000 (Thermo Fisher Scientific), and the concentration was adjusted to 100 ng / μL.

  First, a normal PCR product was obtained for the obtained DNA. Specifically, a normal PCR product was obtained by carrying out 40 normal PCR cycles with the reaction solution composition shown in Table 7 using the primer set shown in Table 3.

  Next, a COLD-PCR product was obtained for the obtained DNA. Specifically, a primer set without GC-Clamp shown in Table 5 was used, and 40 normal PCR cycles were performed with the reaction solution composition shown in Table 7, followed by NucleoSpin (registered trademark) Extract II (MACHEREY-NAGEL). After purification, the obtained amplification product was adjusted to 1 ng / μL. The amplified product is Using the double-stranded RNA for suppressing cerevisiae, the primer set shown in Table 5 was used, and 40 COLD-PCR cycles were performed with the reaction solution composition shown in Table 6. The amplification product was again purified by NucleoSpin (registered trademark) Extract II (MACHEREY-NAGEL), and then the concentration was measured and adjusted to 1 ng / μL. Finally, for the analysis by the DGGE method, as described above, using the primer set described in Table 3, the normal PCR cycle was performed for 40 cycles with the reaction solution composition described in Table 2, and the PCR product (COLD- PCR product) was prepared.

  Each obtained amplification product was electrophoresed by DGGE method on a denaturant gradient gel of 40% to 55%. The results are shown in FIG. Lanes 1 and 2 use DNA obtained from the sample immediately after yeast addition on the day of red wine fermentation as a template, and Lanes 3 and 4 use DNA obtained from the sample on day 8 of red wine fermentation. Lanes 1 and 3 show normal PCR products, and lanes 2 and 4 show COLD-PCR products. When DNA extracted from the sample on the 8th day from the start of fermentation is used as a template (comparison between lanes 3 and 4), COLD-PCR using double-stranded RNA is carried out to detect strongly by adding yeast at the start of wine fermentation. S. that had been done. cerevisiae-derived bands disappeared, indicating that other bands could be detected.

Example 4
S. cerevisiae and S. cerevisiae. Using pombe genome DNA, it was examined whether the technique of the present invention can be applied to long DNA fragments.

  Specifically, the S.I. cerevisiae and S. cerevisiae. A normal PCR product was obtained by carrying out 40 normal PCR cycles with the reaction solution composition shown in Table 7 using the primer set shown in Table 8 for the mixture of pombe genome DNA. Thereby, an amplification product of about 610 to 620 bases is obtained.

  Separately, S.M. cerevisiae genome DNA was subjected to normal PCR using the primer set described in Table 9 and the reaction solution composition shown in Table 7, and double-stranded RNA corresponding to this example was prepared in the same manner as in Example 1. did.

  The concentration of the normal PCR product prepared above was adjusted to 1 ng / μL. COLD-PCR was carried out using the newly prepared double-stranded RNA to suppress the amplification of cerevisiae with the reaction solution composition shown in Table 10.

  Since it becomes difficult to obtain a clear band by DGGE as the chain becomes longer, in this example, S.I. cerevisiae has a KpnI cleavage site in its internal sequence, and S. cerevisiae Pombe does not have a KpnI cleavage site, and whether or not it is cleaved by KpnI. cerevisiae or S. cerevisiae. It was decided to determine the pombe. That is, amplification products obtained by normal PCR have almost the same base length and are difficult to separate by agarose gel electrophoresis. cerevisiae and S. cerevisiae. Both bands of pombe appear as bands at approximately the same position. However, by cutting with KpnI, S. pombe is in the same position as before, S.P. In cerevisiae, a band is detected at a shorter position.

  Specifically, KpnI was manufactured by Takara Bio Inc., and was used at 10 U per 20 μL of a reaction solution containing 1 × L buffer, and the treatment was performed at 37 ° C. for 1 hour. The result of electrophoresis is shown in FIG. When not treated with KpnI, bands of the same length were detected in lane 2 (normal PCR product) and lane 3 (COLD-PCR product of the present invention). cerevisiae and S. cerevisiae. It can be seen that the pombe band is amplified. When lane 4 (ordinary PCR product) and lane 5 (COLD-PCR product of the present invention) are compared, a short band is detected in lane 3 by KpnI treatment. cerevisiae band, and the remaining band is S. cerevisiae. In lane 4, since a short fragment is no longer detected by KpnI treatment, almost all of the band is S.Pombe. It was considered a pombe. Therefore, it was suggested that the selective suppression by this method can be applied to a long-chain DNA of about 600 bases.

Example 5
Detection of single nucleotide polymorphism When it is desired to amplify a sequence containing a single nucleotide polymorphism preferentially over the wild type by the method of the present invention, it should be noted that the difference in Tm value is very small. That is, it is expected that selective amplification cannot be performed even if the Tm value calculated as a theoretical value is used. Therefore, it is desirable to measure an accurate Tm value in advance. As an example, a solution in which primers and enzymes are removed from a PCR reaction solution to be actually used is prepared, and accurate Tm values are calculated using SYBR GREEN I and Real-Time PCR, and DNA: RNA containing mismatches By using the temperature at which the difference between the dissociation of the hybrid and the dissociation of the perfect match DNA: RNA hybrid is the largest as the temperature of the heat denaturation II, it is possible to preferentially amplify the mutant having the mismatch. Therefore, although it is necessary to accurately measure the Tm value and set the temperature of the heat denaturation II, in other respects, it is possible to perform under the same conditions as when performing other polymorphism analysis and microflora analysis. Is possible.

  According to the nucleic acid amplification method of the present invention, amplification of nucleic acid fragments having an arbitrary sequence can be suppressed, and other nucleic acid fragments can be amplified with high efficiency. Useful in related industrial fields.

Sequence number 1 of a sequence table is a base sequence of NL1 primer with GC-Clamp.
Sequence number 2 of a sequence table is a base sequence of LS2 primer.
Sequence number 3 of a sequence table is a base sequence of NL1 primer with a T7 promoter.
Sequence number 4 of a sequence table is a base sequence of LS2 primer with a T7 promoter.
Sequence number 5 of a sequence table is a base sequence of NL1 primer without GC-Clamp.
Sequence number 6 of a sequence table is a base sequence of NL4 primer.
Sequence number 7 of a sequence table is a base sequence of NL4 primer with a T7 promoter.

Claims (5)

DNA was mixed with double-stranded RNA having homologous nucleotide sequence to the sample and the DNA containing two or more DNA (A) comprising (A), after hybridized by thermal denaturation, the DNA A temperature at which hybridization between the DNA other than ( A ) and the RNA is dissociated and the hybridization between the DNA ( A ) and the RNA is maintained, and then the dissociated DNA is amplified. A method for amplifying DNA . The double-stranded RNA is obtained using as a template a PCR product obtained using a universal primer having the sequence of a Small-SubUnit rRNA or Large-SubUnit rRNA gene. DNA amplification method. A method for detecting a single nucleotide polymorphism, comprising detecting a gene polymorphism in a sample using the DNA amplification method according to claim 1. A method for analyzing a microflora, wherein the DNA derived from microorganisms in a sample is detected using the DNA amplification method according to claim 1. A method for improving the amplification efficiency of a low-content DNA fragment contained in a sample, using the DNA amplification method according to claim 1 or 2.