JP2009225787A - Method for identifying unidentified single base substitution type polynucleotide and primer pair - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and rapidly identifying an unidentified single base substitution type polynucleotide. <P>SOLUTION: The method for identifying an unidentified single base substitution type polynucleotide includes identifying whether a base of template polynucleotide (TP) corresponding to a base of 3' terminal of a forward primer (FP) and/or a base of (TP) corresponding to a base of 3' terminal of a reverse primer (RP) is a single base substitution type polynucleotide or not. The method includes a step of carrying out PCR by using a primer pair composed of FP in which 3' terminal is complemented with TP and first or second adjacent base of 3' terminal is not complemented with a base of TP and RP in which 3' terminal is complemented with TP and first or second adjacent base of 3' terminal is not complemented with a base of TP, a step of determining whether amplification of polynucleotide exists or not and a step of identifying a base sequence of unidentified single base substituted polynucleotide by specifying TP corresponding to a primer pair from a primer pair corresponding to the amplified polynucleotide. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an identification method and a primer pair for identifying an unidentified single nucleotide substitution polynucleotide.

  In order to distinguish one base difference, nucleic acid amplification reaction is carried out on a microfluidic device using a primer containing a base mismatched with the template polynucleotide within 5 bases from the 3 ′ terminal base, and whether or not amplification has occurred A method for determining the presence or absence of a single nucleotide polymorphism or mutation of a polynucleotide is known (Patent Document 1).

JP 2005-168350 A

When a conventional determination method is used, strict PCR conditions must be set for each template polynucleotide, and no general-purpose technique has been achieved.
An object of the present invention is to provide a method for identifying an unidentified single nucleotide substitution polynucleotide in a simple and rapid manner and a PCR primer pair optimal for this method.

The identification method of the present invention is characterized by the base (TP1) of the template polynucleotide (TP) corresponding to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the 3 ′ end of the reverse primer (RP). A method for identifying an unidentified single base substitution polynucleotide for identifying whether or not a base (TP3) of a template polynucleotide (TP) corresponding to a base (SB2) is a single base substitution polynucleotide, ,
The base at the 3 ′ end (SB1) is a base complementary to the base (TP1) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB1) are the base of the template polynucleotide (TP). A forward primer (FP) that is a base that is not complementary to the base (TP2);
The base at the 3 ′ end (SB2) is a base complementary to the base (TP3) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB2) are the base of the template polynucleotide (TP). PCR step (1) in which PCR is performed using a primer pair consisting of a base (TP4) and a reverse primer (RP) which is a base that is not complementary;
A determination step (2) for determining the presence or absence of amplification of a polynucleotide by PCR; and by identifying a template polynucleotide corresponding to the primer pair from the primer pair corresponding to the amplified polynucleotide, unidentified single base substitution The gist of the invention is that it has an identification step (3) for identifying the base sequence of the type polynucleotide.

The feature of the PCR primer pair of the present invention is that the base (TP1) of the template polynucleotide (TP) corresponding to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the 3 ′ of the reverse primer (RP) From the forward primer (FP) and reverse primer (RP) for identifying whether or not the base (TP3) of the template polynucleotide (TP) corresponding to the terminal base (SB2) is a single base-substituted polynucleotide Become
The base at the 3 ′ end (SB1) is a base complementary to the base (TP1) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB1) are the base of the template polynucleotide (TP). A forward primer (FP) that is a base that is not complementary to the base (TP2);
The base at the 3 ′ end (SB2) is a base complementary to the base (TP3) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB2) are the base of the template polynucleotide (TP). The gist is that the base consists of a base (TP4) and a reverse primer (RP) that is not a complementary base.

  According to the identification method of the present invention, an unidentified single base substitution polynucleotide can be identified easily and rapidly. Further, when the PCR primer pair of the present invention is used, an unidentified single base substitution polynucleotide can be identified easily and rapidly. Moreover, when the identification method of the present invention is applied to each of two pairs of chromosomes, it can be determined whether the type of single-base substitution is homozygous or heterozygous.

Therefore, when the identification method of the present invention and the PCR primer of the present invention are used, it is not necessary to set strict PCR conditions depending on the template polynucleotide, and it can be widely applied as a general-purpose technique.
In addition, when the template polynucleotide (TP) is a pathogenic microbial DNA, rapid medical treatment is possible, which can greatly contribute to lifesaving.
Further, when the template polynucleotide (TP) is human genomic DNA, the correlation between DNA and disease, drug metabolism or drug reactivity can be elucidated, so that it can greatly contribute to the medical field such as gene diagnosis.

  In the identification method of the present invention, the base (TP1) of the template polynucleotide (TP) corresponding to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the base at the 3 ′ end of the reverse primer (RP) ( A method for identifying an unidentified single base substitution polynucleotide for identifying whether or not the base (TP3) of the template polynucleotide (TP) corresponding to SB2) is a single base substitution polynucleotide, Is a method for identifying a single nucleotide polymorphism or mutation.

  In the present invention, PCR means a polymerase chain reaction (nucleic acid amplification reaction). The base means adenine, thymine, cytosine, guanine or uracil.

  The template polynucleotide is obtained by polymerizing a plurality of nucleotides (nucleic acids), and the number of nucleotides is not particularly limited, but is preferably at least 20, more preferably at least 30, particularly preferably at least 40, most preferably. Is at least 50.

The template polynucleotide may be derived from a living body such as whole blood, serum, fecal matter or urine, or derived from a cell tissue or cell culture such as an animal, plant or virus, and may be a beverage or canned food. It may be derived from processed foods such as
Of the template polynucleotides, DNA is preferable, and human genomic DNA and pathogenic microbial DNA are more preferable (for example, viruses, tuberculosis, cholera, botulus, typhoid, and BSE).

  The template polynucleotide has a known base sequence and corresponds to all or part of the base sequence of the single-base substitution polynucleotide to be identified. Then, a plurality of types of forward primer (FP) and reverse primer (RP) corresponding to the template polynucleotide are synthesized in advance by a known method, and these are used in the PCR step (1).

  The forward primer (FP) is a base in which the base (SB1) at the 3 ′ end is complementary to the base (TP1) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB1) is the template. It is a base that is not complementary to the base (TP2) of the polynucleotide (TP).

  In the reverse primer (RP), the base at the 3 ′ end (SB2) is complementary to the base (TP3) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB2) are It is a base that does not complement the base (TP4) of the template polynucleotide (TP).

  The number of bases of the forward primer (FP) and the reverse primer (RP) is not particularly limited, but is preferably 10 to 50 bases, more preferably 15 to 40 bases, particularly preferably 20 from the viewpoint of PCR reaction efficiency and the like. ~ 35 bases.

  The forward primer (FP) and the reverse primer (RP) may have the same base sequence or different base sequences. The forward primer (FP) and the reverse primer (RP) are preferably different from each other.

  The forward primer (FP) and the reverse primer (RP) anneal to the template polynucleotide, and the unidentified single base substitution polynucleotide is complementary to the base (SB1) at the 3 ′ end of the forward primer (FP), and / or Alternatively, when complementary to the 3 ′ terminal base (SB2) of the reverse primer (RP), the PCR extension reaction proceeds.

  On the other hand, the forward primer (FP) and the reverse primer (RP) anneal to the template polynucleotide, and the unidentified single base substitution polynucleotide does not complement the 3 ′ terminal base (SB1) of the forward primer (FP). And when it does not complement the 3 ′ terminal base (SB2) of the reverse primer (RP), the PCR extension reaction does not proceed.

  That is, when the unidentified single nucleotide substitution-type polynucleotide is complementary to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the base (SB2) at the 3 ′ end of the reverse primer (RP), PCR When the extension reaction proceeds and the unidentified single nucleotide substitution-type polynucleotide does not complement the 3 ′ terminal base (SB1) and the 3 ′ terminal base (SB2), the PCR extension reaction does not proceed.

  In a preferred embodiment of the present invention, the forward primer (FP) and the reverse primer (RP) anneal to the template polynucleotide, and the unidentified single base substitution polynucleotide is the base (SB1) at the 3 ′ end of the forward primer (FP). ) And the 3 ′ terminal base (SB2) of the reverse primer (RP), the PCR extension reaction proceeds.

  On the other hand, the forward primer (FP) and the reverse primer (RP) anneal to the template polynucleotide, and the unidentified single base substitution polynucleotide does not complement the 3 ′ terminal base (SB1) of the forward primer (FP). And / or the PCR extension reaction does not proceed unless it is complementary to the base (SB2) at the 3 ′ end of the reverse primer (RP).

  That is, when the unidentified single nucleotide substitution-type polynucleotide complements the 3 ′ terminal base (SB1) of the forward primer (FP) and the 3 ′ terminal base (SB2) of the reverse primer (RP), the PCR extension reaction When the unidentified single nucleotide substitution-type polynucleotide does not complement the 3 ′ terminal base (SB1) and / or the 3 ′ terminal base (SB2), the PCR extension reaction does not proceed.

In conventional methods for determining the presence or absence of single nucleotide polymorphisms or mutations in polynucleotides, only forward primers are devised, and reverse primers use a common primer. However, even though the nucleotide sequences are not completely complementary, the PCR extension reaction often proceeds.
On the other hand, when the identification method of the present invention is applied, when the unidentified single nucleotide substitution-type polynucleotide and the base at the 3 ′ end of the forward primer (FP) and / or the reverse primer (RP) are complementary, As long as the PCR extension reaction proceeds. In this case, the PCR extension reaction proceeds even though one base of the forward primer (FP) and / or one base of the reverse primer (RP) is not complementary. Therefore, when the identification method of the present invention is applied, unidentified single nucleotide substitution-type polynucleotides can be identified extremely accurately.

  In a preferred embodiment of the present invention, when the identification method of the present invention is applied, an unidentified single nucleotide substitution-type polynucleotide is complementary to the bases at both the 3 ′ end of the forward primer (FP) and the reverse primer (RP). Only in some cases, the PCR extension reaction proceeds. In this case, the PCR extension reaction proceeds even though one base of the forward primer (FP) and one base of the reverse primer (RP) are not complementary. Therefore, when the identification method of the present invention is applied, unidentified single nucleotide substitution-type polynucleotides can be identified extremely accurately.

  The forward primer (FP) and the reverse primer (RP) are usually used in equal amounts, but asymmetric PCR using a large amount of one of these can also be applied.

  The reagent and apparatus used in the PCR step (1) are not particularly limited, and ordinary reagents and apparatuses can be applied.

  Any polymerase can be used as long as it is a reverse transcriptase or an enzyme capable of amplifying nucleic acid, but a thermostable polymerase is preferred.

  In the PCR step (1), a microfluidic device (eg, Patent Document 1) or a plate having a plurality of tubes (eg, Japanese Patent Application Laid-Open No. 2003-102477) is used from the viewpoint of rapidity and simplicity (automation). Preferably it is done.

  Before the PCR step (1), it is preferable to have a PCR step (4) for pre-amplifying an unidentified single nucleotide substitution polynucleotide using a common primer (CP) common to the template polynucleotide. By performing the PCR step (4), a sufficient measurement sample for identification can be secured by the PCR step (4) even if the amount of unidentified mono-substituted polynucleotide is very small. Moreover, when the polynucleotide which does not need to be identified is contained, only the polynucleotide of identification object can be increased by amplification reaction, and the polynucleotide which does not need to be identified can be excluded.

  The common primer (CP) can be used without limitation as long as it is a common primer common to the template polynucleotide. For example, when the template polynucleotide is pathogenic microbial DNA, a common primer corresponding to 16S ribosomal RNA is preferred.

  In the determination step (2) for determining the presence or absence of amplification by PCR, the presence or absence of amplification can be determined by a known method (Patent Document 1 and Japanese Patent Application Laid-Open No. 2003-102477). For example, the presence or absence of amplification by PCR can be determined by a gel electrophoresis method, a fluorescence labeling method, or the like. Among these, a method using a fluorescent label is preferable from the viewpoint of rapidity and simplicity (automation).

  In the identification step (3), the template polynucleotide corresponding to the primer pair is identified from the primer pair corresponding to the polynucleotide confirmed to be amplified by PCR in the determination step (2), and then the base sequence of the template polynucleotide is determined. From this, the base sequence of the unidentified single base substitution polynucleotide is identified (the base sequence of the unidentified single base substitution polynucleotide includes the base sequence of the template polynucleotide).

  Any or all of the PCR step (1), the determination step (2) and the identification step (3) can be automated by mechatronics technology, etc., from the viewpoint of accuracy, rapidity, simplicity (automation), etc. It is preferable to automate all of the PCR step (1), the determination step (2) and the identification step (3).

  The PCR primer pair of the present invention comprises a forward primer (FP) and a reverse primer (RP) and is suitable for the above identification method, but can also be applied to other identification methods. In addition, the PCR primer pair of the present invention may be a mixture of a forward primer (FP) and a reverse primer (RP), or a forward primer (FP) and a reverse primer (RP). Moreover, it may be in the form of a solution or in a dry state. That is, there is no limitation on the form and the like as long as they are used together in the PCR reaction.

  It is preferable that the primer pair of the present invention is different in forward primer (FP) and reverse primer (RP). In the primer pair of the present invention, the corresponding template polynucleotide (TP) is preferably pathogenic microbial DNA or human genomic DNA.

<Example 1>
27 bacterial 16S rRNA nucleotide sequences shown in Table 1 were obtained from the National Center for Biotechnology Information (NCBI) only for bacterial food poisoning bacteria, and ClustalW (National Genetics) was obtained for the 27 bacterial sequences. Homologous sequences and non-homologous sequences were identified by alignment using a research institute website.

  The homologous sequence was used to design the next common primer (cp1) (SEQ ID NO: 1 and 2) that amplifies the 16S rRNA region of the pathogen.

CF: 5'-gaacgctggc ggcaggccta a-3 '
CR: 5'-ggtgtgacgg gcggtgagta caa-3 '

  Using a non-homologous sequence in the 16S rRNA region of a pathogenic bacterium, a primer that can specifically amplify against Escherichia coli (hereinafter abbreviated as E. coli) JM109 genomic DNA, and a non-homologous part as a primer 3 ' The next forward primer and reverse primer were designed by mismatching one or two adjacent bases of the 3 ′ terminal base (SB1) as the terminal base (the part indicated in capital letters was mismatched) .) In the non-homologous part, the non-homologous sequence had only one base.

<Forward primer (SEQ ID NO: 3 to 8)>
A1: 5'-gtcga acggt aacag gaaCa-3 '
A2: 5'-gtcga acggt aacag gaaTa-3 '
A3: 5'-gtcga acggt aacag gaGga-3 '
A4: 5'-gtcga acggt aacag gaCga-3 '
B1: 5'-tgctc ttcgc tgacg agtAc-3 '
B2: 5'-tgctc ttcgc tgacg agtCc-3 '

<Reverse primer (SEQ ID NO: 9 to 13)>
C1: 5'-gcaaa ggtat taact ttCct-3 '
D1: 5'-tctgc gggta acgtc aatAa-3 '
D2: 5'-tctgc gggta acgtc aatTa-3 '
D3: 5'-tctgc gggta acgtc aaCga-3 '
E1: 5'-gtccc cctct ttggt cttgc-3 '

  Each of E. coli and Salmonella typhimurium (hereinafter abbreviated as Salmonella) was extracted with nucleic acid, the concentration was adjusted to 1 ng / μl, and a common primer (cp1) (10 pmol / μl) was used as a template polynucleotide. PCR was performed. Next, the presence or absence of amplification of the polynucleotide was determined by electrophoresis (ethyl bromide, UV irradiation) to obtain an amplification product.

PCR was carried out using the amplification product (1 ng / μl) as a template polynucleotide and the primer pairs shown in Table 2 (forward primer and reverse primer solutions, 10 pmol / μl each). Next, the presence or absence of amplification of the polynucleotide was determined by electrophoresis (ethyl bromide, UV irradiation), and the measurement sample extracted from E. coli was amplified, but amplification was observed for the measurement sample extracted from Salmonella. It was confirmed that it was not possible.
The measurement sample, the forward primer, and the reverse primer were each used as a solution of TE Buffer (10 mM Tris-HCl Buffer / 1 mM EDTA (PH7.5)).

The PCR conditions are as follows.
10 × NH ₄ Reaction Buffer ₅ μl (Bioline)
2.5 mM dNTP Mix 4 μl (Bioline)
50 mM MgCl ₂ 5 μl
Measurement sample 1μl
1 μl of forward primer
1 μl reverse primer
Thermostable DNA polymerase Bio Taq (5 U / μl) 0.5 μl (Bioline)
35.5 μl of sterile distilled water
Reaction time 30 seconds Reaction temperature 95 ° C
Denaturation time 10 seconds Denaturation temperature 95 ° C
Annealing time 20 seconds Annealing temperature 55 ° C
Extension time 1 minute (After 30 cycles, extension reaction was performed for 2 minutes)
Elongation temperature 72 ° C
30 cycles

<Example 2>
Using a non-homologous sequence in the 16S rRNA region of a pathogenic bacterium, a primer that can be specifically amplified to Salmonella genomic DNA is converted into a base at the 3 ′ end of the primer using the non-homologous part as the base at the 3 ′ end of the primer ( A forward primer and a reverse primer were designed in the same manner as in Example 1 by mismatching one or two adjacent bases of SB1). In the non-homologous part, the non-homologous sequence had only one base.

<Forward primer (SEQ ID NO: 14)>
F1: 5'-gctga cgagt gccgg acAgt-3 '

<Reverse primer (SEQ ID NO: 15)>
G1: 5'-tgagc ccggg gattt caAac-3 '

  In the same manner as in Example 1, nucleic acids were extracted from E. coli and Salmonella, the concentration was adjusted to 1 ng / μl, and PCR was performed using a common primer (cp1) (10 pmol / μl) as a template polynucleotide. went. Next, the presence or absence of amplification of the polynucleotide was determined by electrophoresis (ethyl bromide, UV irradiation) to obtain an amplification product.

  In the same manner as in Example 1, using the amplification product (1 ng / μl) as a template polynucleotide and a primer pair (19) (a solution of forward primer (F1) and reverse primer (G1) (each 10 pmol / μl), Next, the presence or absence of amplification of the polynucleotide was determined by electrophoresis (ethyl bromide, UV irradiation), and the measurement sample extracted from Salmonella was amplified, but the measurement sample extracted from E. coli It was confirmed that no amplification was observed.

<Example 3>
Each of E. coli, Salmonella, Vibrio cholerae and Clostridium botulinum was subjected to nucleic acid extraction, the concentration was adjusted to 1 ng / μl, and PCR was performed using a common primer (cp1) (10 pmol / μl) as a template polynucleotide. . Next, the presence or absence of amplification of the polynucleotide was determined by electrophoresis (ethyl bromide, UV irradiation) to obtain an amplification product. Each amplification product was mixed, adjusted to 1 ng / μl, and used as a measurement sample. For the measurement sample, a mixed amplification product was obtained in the same manner as in Example 1 using the common primer (CF / CR).

Subsequently, PCR was performed on the mixed amplification product in the same manner as in Example 1 using the primer pair (1) shown in Table 2 and the primer pair (19) (F1 / G1) obtained in Example 2.
Next, when the presence or absence of amplification of the polynucleotide was determined, amplification was performed when the primer pair (1) was used, and amplification was not observed when the primer pair (19) was used.
Therefore, from the primer pair (1), the measurement sample was identified as a polynucleotide derived from E. coli.

<Example 4>
For the mixed amplification product prepared in Example 3, PCR was performed in the same manner as in Example 1 using the primer pair (2) shown in Table 2 and the primer pair (19) (F1 / G1) obtained in Example 2. Went.
Next, when the presence or absence of amplification of the polynucleotide was determined, amplification was performed when the primer pair (19) was used, and amplification was not observed when the primer pair (1) was used.
Therefore, from the primer pair (19), the measurement sample was identified as a polynucleotide derived from Salmonella.

<Example 5>
Six types of osteoporosis-related polymorphic genes (3 types of ER gene polymorphism and 3 types of VDR gene polymorphism) were obtained from Nipro Research Institute. These DNA fragments are double-stranded DNA (1st PCR product) amplified by the PCR reaction, and contain one genetic polymorphism on the gene sequence. The ER gene polymorphism is G / G type, G / A type, and A / A type. The VDR gene polymorphism is T / T type, T / C type, and C / C type. It is a kind.

  For six types of osteoporosis-related polymorphic gene sequences, two types of primers (forward primer and reverse primer) were designed such that the second or third base sequence from the 3 'end mismatched. The reverse primer was designed so that the 3'-end primer sequence corresponds to the position of the gene polymorphism on the genome. The forward primer was designed so that the sequence at the 3 'end would be a sequence homologous to the sequence of the 1st PCR product. A PCR reaction was performed using these two types of primers, and the gene sequence of each type of osteoporosis was determined depending on whether or not amplification was possible.

The base sequences of the forward primer and the reverse primer are shown below.
The part written in capital letters is the part that was arbitrarily mismatched. The 3 ′ end of the forward primer is a sequence homologous to the 1st PCR product, and the 3 ′ end sequence of the reverse primer is a sequence homologous or non-homologous to the position of the gene polymorphism on the genome.

<Forward primer (SEQ ID NO: 16 to 18)>
ER gene:
EF4: 5'-ctgtg ttgtc catca gtAca-3 '
VDR gene:
VF5: 5'- ctata taggc agaac caCct-3 '
VF6: 5'- ctata taggc agaac caGct-3 '

<Reverse primer (SEQ ID NO: 19-22)
ER gene ERa4: 5'-ccaat gctca tccca acAct-3 '
ERg3: 5'-ccaat gctca tccca actTc-3 '
VDR gene:
VRt2: 5'-gggcc acaga caggc ctgAa-3 '
VRc3: 5'-gggcc acaga caggc ctgTg-3 '

The conditions for PCR are as follows.
10 × NH ₄ Reaction Buffer 0.4 μl (Biolines)
2.5 mM dNTPS Mix 1.6 μl (Biolines)
50 mM MgCl ₂ 0.8 μl
Measurement sample (1st PCR product) 0.4 μl
Forward primer 0.4 μl (4 pmol)
Reverse primer 0.4 μl (4 pmol)
Thermostable DNA polymerase Bio Taq (5 U / μl) 0.2 μl
Sterile distilled water 14.2 μl
Total 20μl
PCR reaction conditions:
94 ℃ 5min
94 ° C 30 sec
65 ° C 30 sec
72 ° C 30 sec
72 ° C 7min
Number of cycles 30 or 40 (only when determining VDR gene polymorphism)
Primer combinations are shown in the table below

In Table 3, when specific amplification is seen with the combination of primer pair 1 and no amplification is seen with the combination of primer pair 2, the ER gene polymorphism is A / A type. In addition, when specific amplification is observed in combination of primer pairs 1 and 2, the ER gene polymorphism is G / A type. Moreover, when specific amplification is seen only with the combination of primer pair 2, the ER gene polymorphism is G / G type.
Similarly, when a specific amplification is seen with the combination of primer pair 3 and no amplification is seen with the combination of primer pair 4, the VDR gene polymorphism is T / T type, and the combination of primer pairs 3 and 4 is both When specific amplification is seen, the VDR gene polymorphism is T / C type, and when specific amplification is seen only with the combination of primer pair 4, the VDR gene polymorphism is C / C type.

  Each type of osteoporosis gene 1st PCR product was used as template DNA, diluted 10-fold in concentration, and PCR was performed using the primer pairs shown above. Next, the presence or absence of target sequence amplification was determined by electrophoresis. As a result, both ER genotype and VDR genotype showed amplification specific to each genotype, and genotype could be determined. Further, these results were consistent with the base sequence determined in advance by ABI DNA sequencer.

<Comparative Example 1>
About the mixed amplification product prepared in Example 3, using primer pair (20) (forward primer (A1) and reverse primer (CR), and primer pair (21) (forward primer (F1) and reverse primer (CR)) PCR was performed in the same manner as in Example 1.
Next, when the presence or absence of amplification of the polynucleotide was determined, amplification was confirmed both when the primer pair (20) was used and when the primer pair (21) was used.

<Comparative Example 2>
For the mixed amplification product prepared in Example 3, using primer pair (22) (forward primer (A2) and reverse primer (CR), and primer pair (21) (forward primer (F1) and reverse primer (CR)) PCR was performed in the same manner as in Example 1.
Next, when the presence or absence of amplification of the polynucleotide was determined, amplification was confirmed both when the primer pair (22) was used and when the primer pair (21) was used.

Claims (11)

Template polynucleotide corresponding to the base (TP1) of the template polynucleotide (TP) corresponding to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the base (SB2) at the 3 ′ end of the reverse primer (RP) A method for identifying an unidentified single base substitution polynucleotide for identifying whether or not a base (TP3) of a nucleotide (TP) is a single base substitution,
The base at the 3 ′ end (SB1) is a base complementary to the base (TP1) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB1) are the base of the template polynucleotide (TP). A forward primer (FP) that is a base that is not complementary to the base (TP2);
The base at the 3 ′ end (SB2) is a base complementary to the base (TP3) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB2) are the base of the template polynucleotide (TP). PCR step (1) in which PCR is performed using a primer pair consisting of a base (TP4) and a reverse primer (RP) which is a base that is not complementary;
A determination step (2) for determining the presence or absence of amplification of a polynucleotide by PCR; and by identifying a template polynucleotide corresponding to the primer pair from the primer pair corresponding to the amplified polynucleotide, unidentified single base substitution The identification method characterized by having the identification process (3) which identifies the base sequence of a type | mold polynucleotide. Template polynucleotide corresponding to base (TP1) of template polynucleotide (TP) corresponding to base (SB1) at 3 ′ end of forward primer (FP) and base (SB2) at 3 ′ end of reverse primer (RP) ( The identification method according to claim 1, which is a method for identifying an unidentified single base substitution polynucleotide for identifying whether or not the base (TP3) of TP) is a single base substitution polynucleotide. The identification method according to claim 1 or 2, wherein the PCR step (1) is performed using a microfluidic device or a plate having a plurality of tubes. The identification method according to claim 1, wherein the PCR forward primer (FP) and the PCR reverse primer (RP) are different from each other. The identification method according to any one of claims 1 to 4, wherein the template polynucleotide (TP) is pathogenic microbial DNA or human genomic DNA. The identification according to any one of claims 1 to 5, further comprising a PCR step (4) for preamplifying an unidentified single nucleotide substitution polynucleotide using a common primer (CP) common to the template polynucleotide (TP). Method. The identification method according to claim 6, wherein the common primer (CP) is a common primer corresponding to 16S ribosomal RNA. Template polynucleotide corresponding to the base (TP1) of the template polynucleotide (TP) corresponding to the base (SB1) at the 3 ′ end of the forward primer (FP) and / or the base (SB2) at the 3 ′ end of the reverse primer (RP) Consisting of a forward primer (FP) and a reverse primer (RP) for identifying whether the nucleotide (TP3) of the nucleotide (TP) is a polynucleotide having a single base substitution,
The base at the 3 ′ end (SB1) is a base complementary to the base (TP1) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB1) are the base of the template polynucleotide (TP). A forward primer (FP) that is a base that is not complementary to the base (TP2);
The base at the 3 ′ end (SB2) is a base complementary to the base (TP3) of the template polynucleotide (TP), and one or two adjacent bases of this base (SB2) are the base of the template polynucleotide (TP). A primer pair for PCR comprising a base (TP4) and a reverse primer (RP) which is a base that is not complementary. Template polynucleotide corresponding to base (TP1) of template polynucleotide (TP) corresponding to base (SB1) at 3 ′ end of forward primer (FP) and base (SB2) at 3 ′ end of reverse primer (RP) ( The primer pair for PCR according to claim 8, comprising a forward primer (FP) and a reverse primer (RP) for identifying whether or not the base (TP3) of TP) is a single base-substituted polynucleotide. The primer pair according to claim 8 or 9, wherein the forward primer (FP) and the reverse primer (RP) are different from each other. The primer pair according to any one of claims 8 to 10, wherein the template polynucleotide (TP) is pathogenic microbial DNA or human genomic DNA.