JP4020845B2 - Base mutation analysis method - Google Patents

Description

  Genetic analysis of living organisms is widely used in relation to the risk level of diseases, diagnosis, diagnosis, presentation of treatment methods, and the like. For example, it is possible to predict the degree of risk for a disease through mutation analysis for a specific gene of a specific person, and to induce prevention efforts. Or, it is possible to induce effective treatment by investigating whether or not the virus is resistant to the therapeutic drug through genetic mutation analysis of another living organism that causes hepatitis in the living organism, such as a virus. be able to. The present invention relates to a method for analyzing a gene of an organism, and more particularly to a method for investigating a genetic variation.

  As human genome maps have been completed, the possibility of measuring the risk to a disease, diagnosing or prognosing the disease, and predicting a response to a drug is presented. When genome sequence analysis is performed on a large number of individuals, the position on the chromosome showing diversity among individuals appears. This is called SNP (single nucleotide polymorphism). SNP is a mutation that appears at a certain frequency or more in a base sequence arranged on a chromosome of a living organism, and in the case of human beings, it is said that it appears with a probability of about 1 for every 1,000 bases. When considering the size of the human chromosome, there are millions of SNPs. SNP is considered as a means of explaining the phenotypic differences between individuals, and can be used to determine the cause of the disease and prevent or treat the disease.

  The SNPs discovered by the Human Genome Project only show the fact that they show diversity among individuals, and do not tell how the diversity is related to disease. In order to clarify the relationship between SNP and disease, a process of comparative analysis (SNP scoring) of mutant forms of SNP appearing in normal persons and patients is necessary. In order to clearly determine the relationship between SNPs and diseases, not only a large number of SNPs must be analyzed, but also mutations must be able to be analyzed without error.

  SNP scoring methods include DNA sequencing, PCR-SSCP (Polymerase chain reaction-Single stranded conformation polymorphism), allele specific hybridization, oligo-ligation (oligoligation) method, mini There are methods such as sequencing by mini-sequencing and enzymatic cleavage. Although a method using a DNA chip has been introduced, in principle, there is no difference from allele-specific hybridization, and only a difference is made in the stationary phase to which an oligonucleotide probe or the like is attached.

  There are Maxam-Gilbert method and Sanger method for DNA sequencing, but the latter method is mainly used at present. This method is a method for clarifying the base sequence of the entire gene or a part of the gene rather than investigating only the possibility of genetic mutation at a specific position. Since it is also possible to confirm whether or not a genetic mutation at a specific position is possible, it can be used for SNP scoring. However, it is inefficient not to confirm only the mutation of the SNP to be investigated, but to read together the surrounding base sequences that do not need to be investigated.

PCR-SSCP (Orita, M. et.al, Genomics, 1989, 5: 8874-8879) amplified a sequence containing SNPs to be analyzed by PCR, separated into each chain, and then polyacrylamide gel. Perform electrophoresis at Since the secondary structure of the DNA chain differs depending on the difference of one base, the possibility of base mutation is investigated based on the difference in electrophoretic migration speed due to this difference.
Allele-specific hybridization is a method in which sample DNA labeled with a radioisotope or the like is hybridized to a probe attached to a nylon filter, etc., but the possibility of base mutation is investigated by adjusting the hybridization conditions such as temperature. It is.
In the oligoligation method (Nucleic Acid Research 24, 3728, 1996), the reaction was performed under the condition that ligation (ligation) does not occur in a state that is not complementary to the template DNA, and it was confirmed whether the ligation was advanced. This is a method for investigating base mutations.
Mini-sequencing (Genome Research 7: 606, 1997) detects the presence of a single base that has been polymerized under the condition that only one base is polymerized at the position to be examined for mutation. It was developed for SNP scoring in a way that was devised to have different responses.

PCR-SSCP, allele-specific hybridization, oligoligation method, etc. are inconvenient to analyze a large amount of sample using polyacrylamide gel, or the error that occurs because the probe cannot bind to the desired position cannot be confirmed. There is a problem.
Mini-sequencing is a technique developed for SNP scoring, which is easy to analyze and advantageous for analyzing a large number of samples. However, it still has the disadvantage that erroneous results due to errors cannot be confirmed. There is a limitation that it is impossible to find deletions and insertions.

  One of the techniques developed for SNP scoring is the enzyme cleavage method (WO 01/90419). In this method, the base sequence to be analyzed is amplified by the same method as PCR, but the amplified product contains a base sequence that can be cleaved or recognized by two restriction enzymes. In this method, the amplified product is cleaved with two types of restriction enzymes, and the molecular weight of a fragment produced is measured to determine whether or not a base mutation is possible. This analysis method has an advantage that the analysis stage is simple and quick because a fragment is prepared by restriction enzyme reaction immediately after amplification of the gene by PCR and the molecular weight is measured by mass spectrometry or the like. However, the enzymatic cleavage method described in WO 01/90419 still cannot confirm a wrong analysis due to errors. For example, when a primer binds to an undesired position during PCR, an erroneous analysis result may occur, but there is a problem that it cannot be confirmed whether or not the primer has bound to an undesired position. For example, the primer used to investigate the base mutation of CYP2C9 may bind to CYP2C8. In this case, the fact that the primer bound to CYP2C8 other than CYP2C9 cannot be confirmed, so whether or not an error has occurred. It is difficult to confirm. Furthermore, although a mutation in which one base is replaced with another base can be detected, there is a problem that a mutation due to deletion or insertion of a base cannot be detected. Furthermore, there is a problem that two or more adjacent base mutations cannot be simultaneously measured.

WO 01/90419 Orita, M.A. et.al, Genomics, 1989, 5: 8874-8879 Nucleic Acid Research 24, 3728, 1996 Genome Research 7: 606, 1997

  The present invention solves the above-mentioned problems and has been devised based on the above-mentioned need, and an object of the present invention is to provide a method capable of accurately and efficiently investigating a genetic variation in a living organism. .

The present invention provides a method for accurately and efficiently analyzing a genetic variation of an organism.
Briefly describing the present invention, the present invention can easily and quickly investigate base mutations in a large number of samples, while verifying errors that may be generated by binding of primers to wrong positions. By doing so, it is possible to investigate the exact base mutation, and not only simultaneously investigate the mutation of many adjacent bases within the range of 32 bases, but also to detect genetic mutation due to deletion or insertion. Developed a possible method. In particular, when multiple genotypes exist simultaneously in a living organism, it is possible to distinguish whether nucleotide mutations at different positions are present simultaneously in one genotype or mixed in different genotypes. . For example, humans have two (one pair) chromosomes containing the same genetic information, and if a genetic mutation occurs, all two chromosomes may have a mutation (homo), but only one chromosome It can happen (hetero). When two or more adjacent base mutations are all heterozygous, two base mutations may exist simultaneously in one chromosome, but may exist in different chromosomes. Since the two cases may have different effects on living organisms, it is necessary to distinguish them. In the case of a virus that infects humans, several types of genotypes may exist together. When two or more adjacent base mutations are all heterogeneous, it is necessary to distinguish whether two base mutations exist simultaneously in one genotype or are divided into different genotypes.

  In the present invention, in order to analyze genetic variation, a desired base sequence is amplified so that the resultant product contains a site that can be cleaved by a restriction enzyme. However, the number of bases of a fragment cleaved by a restriction enzyme should be 32 or less. In this method, at least one base is generated by replication of a template that is not a primer, and the amplified product is cleaved with a restriction enzyme, followed by measuring the molecular weight, thereby providing a method for analyzing genetic variation.

That is, the present invention amplifies a specific polynucleotide using a forward primer and a reverse primer, cleaves the amplified specific polynucleotide with a restriction enzyme, and has a mutant sequence having a size of 2 to 32 bases. A method for gene mutation analysis comprising the steps of generating two or more types of single muscle polynucleotide sections comprising: and measuring the molecular weight of the cut sections. In the present invention, one of two or more base mutations different from each other is present only in one single-muscle (single-strand) polynucleotide section, and the other single-muscle nucleotide section has two or more bases. It is preferred to cleave to include all mutations. For example,. . . ATG. . . And A and G are mutant sequences, the first single muscle nucleotide produced by restriction enzyme cleavage contains only A in the two mutant sequences, and the second single muscle nucleotide is This is a case of cutting so as to include all of A and G.
The present invention provides a method for amplifying a desired base sequence to include a site that can be cleaved by a restriction enzyme in order to analyze a genetic variation, but producing a fragment having the structure shown in Table 1 below.

A “restriction enzyme recognition sequence” is a sequence recognized simultaneously or adjacent to different restriction enzymes, and may not match the sequence to be cleaved. For example, Fok1 and BstF5I all recognize the GGATG sequence, but the cleaved positions are next to the 9th / 13th and 2nd / 0th bases from the 3 ′ end of the recognition sequence, respectively. The two restriction enzymes that can be used in the present invention for recognizing restriction enzyme recognition sequences are all restriction enzymes that can be used for the purpose of the present invention, that is, restriction enzymes having the same or different optimum temperatures. Among them, it is more preferable that they have optimum temperatures different from each other, and Fok1, Bbv I, Bsg I, Bcg I, Bpm I, BseR I or Bae I are restriction enzymes having relatively low optimum temperatures. Preferably, BstF5 I, Taq I, BsaB I, Btr I, BstAP I, Fau I, Bcl I, Pci I or Apo I are preferred as restriction enzymes having relatively high optimum temperatures, and Fok1 and BstF5 I pairs are most preferred. .
Restriction enzymes Bae I have 25 ° C., Fok1, Bbv I, Bsg I, Bcg I, Bpm I, BseR I, Mmel I and AvaII have a relatively low optimum temperature of 37 ° C., and BstF5 I and Taq I have 65 C, BsaB I, Btr I and BstAP I have a relatively high optimum temperature of 60 ° C., Fau I has a temperature of 55 ° C., Bcl I, Pci I and Apo I have a relatively high optimum temperature of 50 ° C.

One of the two primers used for PCR amplification is composed of a primer binding sequence 1, a restriction enzyme recognition sequence, and a primer binding sequence 2. The remaining one is composed of a primer binding sequence 3.
A “primer binding sequence” is a sequence that can be complementarily bound to a template nucleic acid, but a restriction enzyme recognition sequence may not be complementary to a nucleic acid. Primer binding sequences 1, 2, and 3 must be at least 4 in order for the primer to bind to the template DNA, so the number of bases must be at least 4 and has a size of 8-30. It is preferable that the number is 8 or more and 30 or less because it binds best with the template DNA. The “pre-mutation sequence” is the sequence of the 5 ′ base mutation to be investigated. The “mutant sequence” is a sequence corresponding to the base mutation to be investigated. Although base substitution, insertion, and deletion may occur, the number of bases is usually one, and there may be two or more. The “post-mutation sequence” is the sequence of 3 ′ of the mutant sequence.

  It is preferable that the pre-mutation sequence and the post-mutation sequence are one or more. The section generated after cleavage of the restriction enzyme must contain a mutated sequence, and the number of bases in the section is preferably 2 to 32. More preferably, it has 12 bases. The reason for numerically limiting the size of the cut section is that when analyzed by mass spectrometry, the analysis result reflects the size of the section with good results. Since the section having a size of more than 32 is too large for investigating the base mutation by calculating the molecular weight using mass spectrometry, the number of bases in the section as described above is preferable. Furthermore, since a section consisting of only one base contains only the mutated sequence, it is not preferable because it makes it difficult to confirm whether the primer is bound at the wrong position. Since the two restriction enzymes recognize the same or adjacent sites, it is preferable that the other restriction enzyme does not act while the reaction of any one of the two restriction enzymes proceeds. In such a case, when the amplified product is cleaved with a restriction enzyme, it can be continuously reacted at different temperatures in view of the optimum temperature of the two restriction enzymes. Alternatively, it is possible to first cleave with one restriction enzyme and then cleave with the remaining enzymes. At this time, the restriction sequence previously used should not remove or destroy the recognition sequence or cleavage position of the second restriction enzyme present in the section containing the mutated sequence.

  The present invention can verify an erroneous analysis due to an error that the existing base mutation analysis method has, and can simultaneously investigate a number of adjacent base mutations within a range of 32 bases. In particular, when there are several types of genotypes of organisms that exhibit base mutations, whether there are base mutations at different positions in one genotype at the same time, or is there a mixture of two or more genotypes? Can be distinguished. Furthermore, genetic mutations due to deletions or insertions can also be measured.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

2741th base mutation of the 4th intron of the human maspin gene 2741st base of the 4th intron of the maspin (serpinb5) gene (rs1509477; chromosome 18th) known as a human cancer metastasis suppressor gene 61001755 base) mutation was investigated.

1. PCR amplification and restriction enzyme cleavage
The template DNA sequence (5 ′ → 3 ′) is as follows.
GTT TCACTTGATAAAGCAAATAAATGCTATTCA cAGCT GCATGAGGCTACACCCTTCTTTTGAAT GCAG ( SEQ ID NO: 1)
The underlined sequence is the site where the following primers 1 and 2 bind. Bases written in lower case are 'mutant sequences' (hereinafter referred to as bases located at the SNP site) .
Primer 1.5'-TCACTTTGAATAAGCAATAAAAggatgGCTATTCA-3 '(34mer) ( SEQ ID NO: 2)
Primer 2.5'-CATTCAAAAGAGGGGTGTAGCCCTCATGC-3 '(28mer) ( SEQ ID NO: 3)
The sequence written in lower case is the recognition sequence for Fok I and BstF5I.
PCR buffer (1x), MgSO ₄ 2 mM, dNTP 200 mM, Platinum Taq Polymerase (Invitrogen, 10966-026) 0.315 U, primers 1 and 2 each 0.5 uM, genomic DNA 36 ng, total reaction volume to 18 μl Match. And PCR reaction is performed on the following conditions.
94 ° C for 2 minutes,
94 ° C 15 seconds 55 ° C 15 seconds 72 ° C 30 seconds (10 cycles)
94 ℃ 15 seconds 60 ℃ 15 seconds 72 ℃ 30 seconds (35cycles)

  Genomic DNA is extracted from blood and purely separated by conventional methods. For example, the 'SDS / protease K' method can be used (Maniatis, Molecular Cloning, A laboratory Manual, Cold Spring Harber Laboratory Press, Cold Spring Harber, 1989) or QIAamp DNA Mini Kit 250 (Qiagen 51106). DNA can be separated from blood. When the DNA concentration is low, the DNA can be concentrated and used by the following method. 1/10 volume of 3M Sodium acetate (pH 5.3) and 2.5 volume of ethanol are added to the DNA solution and gently mixed, and then left at -20 ° C for 1 hour or longer. Centrifuge for 15 minutes at 13000 rpm at 4 ° C. Carefully remove the supernatant, add 70% ethanol, and centrifuge at 13,000 rpm for 10 minutes at 4 ° C. After the ethanol is dried, the desired volume of distilled water is added and dissolved.

The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
TCACTTTGAATAAGCAATAAAAAggatgGC TATTCA [C / T] AGCTGCCATGAGGCTACACCCTTCTTTTGAATG ( SEQ ID NO: 4)
AGTGAACTATTTCGTTATTTTcctac CGATAAGT [G / A] TCGA CGTACTCCGATGTGGAGAAAAAACTTAC ( SEQ ID NO: 5)
The site shown in lower case is a sequence recognized by Fok I and BstF5I, the underlined site is the sequence of a fragment generated by restriction enzyme cleavage, and the base shown in brackets ([]) is SNP. A base located at a site . This reaction was mixed with FokI (NEB R109L) 1U, BstF5I (NEB, V0031L) 1U, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT ( pH 7.9@25° C.) at 25 ° C. The reaction is carried out for 2 hours continuously at 45 ° C. for 2 hours.

  In order to optimize the enzyme reaction, the amplified product was reacted with Fok1 and BstF5I at 25 ° C, 37 ° C, 45 ° C, 55 ° C, and 65 ° C. As a result, in the case of FokI, 70% and 37% at 25 ° C. It was confirmed that the reaction proceeded by 90% or more. In addition, in the case of BstF5I, it was confirmed that the enzyme reaction proceeded at a temperature excluding 25 ° C. Therefore, it is preferable that the reaction is first performed at 25 ° C. at which FokI can act alone and then at 37 ° C. or higher at which BstF5I can react.

2. Purification and desalting It is preferable to measure the molecular weight after pure separation of DNA sections from the solution treated with restriction enzymes. For example, Nucleave Genotyping Kit (Variagenics, USA) can be used. First, 70 μl of 1M TEAA (Triethylammoniumacetate, pH 7.6) is added to the restriction enzyme reaction solution and left for 1 minute. 70 μl of 1M TEAA and 90 μl of the above mixed solution are sequentially put into the sample preparation plate and passed through, and then 85 μl of 0.1M TEAA is completely passed through 5 times. Centrifuge the Sample Preparation Plate at 1000 rpm for 5 minutes. Place the Sample Preparation Plate on the Collection Plate, add 60 μl of 60% isopropanol and let it pass. When the eluate is collected on the collection plate, the collection plate is dried at 115 ° C. for 75 minutes.

3. MALDI-TOF mass spectrometry
After adding 6 μl of MALDI matrix (22.8 mg ammonium citrate, 148.5 mg hydroxypicolinic acid, 1.12 ml acetonitrile, 7.8 ml H ₂ O) to the collection plate, 4 μl of this was added to Anchor of MALDI-TOF (Biflex IV, Bruker). Place on the chip plate. The sample is dried at 37 ° C. for 30 minutes, allowed to cool to room temperature for a while, and then analyzed by MALDI-TOF. The analysis method follows the MALDI-TOF manual.
When the 2741st base of the fourth intron is normal (C / C), the molecular weights of the sections generated after enzymatic cleavage are 215.4D (7mer) and 4078.6 (13mer) D (FIGS. 1 and 2). In the case of heterogeneity (C / T), the molecular weights of the generated sections are 215.4D, 210.4D (more than 7mer), 4078.6D, and 4062.6D (13mer) (FIGS. 3 and 4). On the other hand, when all are mutated to T (T / T), the molecular weights of the sections are 210.4D (7mer) and 4062.6D (13mer) (FIGS. 5 and 6).

Mutation of base 3597 (rs1396782; base 18002611 of chromosome 18) of the fourth intron of the maspin (serpinb5) gene known as a human cancer metastasis suppressor gene
The sequence of Template DNA is as follows.
CTG GAGTATTATCCCTTGCAGGCTTGATATGAAG cTTGAAAT TTCTCCCCAAAGAGATTTAGTATACAGGC AAA ( SEQ ID NO: 6)
The underlined site in the above sequence is the site where the following primers 3 and 4 bind respectively. The base indicated in lower case is the base located at the SNP site .
Primer 3.5 '- GAGTATTATCCTTGCAGGCTTggatgATATGAAG - 3' ( 34mer) ( SEQ ID NO: 7)
Primer 4.5'-GCCTGTTAACTAAATCTCTTTGGGGGAGAA-3 '(29mer) ( SEQ ID NO: 8)
The site indicated in lower case in the above primer is a sequence that does not exist in Template DNA and is a sequence recognized by Fok I and BstF5I. The experimental method including the PCR reaction is the same as in Example 1.

The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
GAGTATTATCCCTTGCAGGCTTTagatgAT ATGAAG [C / T] TTGAAAATTTCTCCCCCAAAGAGATTTAGTATACAGGC ( SEQ ID NO: 9)
CTCATAATAGGAACGTCCGAAcctac TATACTC [G / A] AACT TTAAAAGGGGGTTTCTCTAAACATATTGCTC ( SEQ ID NO: 10)
The site shown in lower case in the above sequence is a restriction enzyme recognition sequence, the underlined site is the sequence of a fragment generated by cleavage of the restriction enzyme, and the base indicated in brackets ([]) is the SNP site. Is the base located at This reaction was mixed with FokI (NEB R109L) 1U, BstF5I (NEB, V0031L) 1U, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT ( pH 7.9@25° C.) at 25 ° C. The reaction is carried out for 2 hours continuously at 45 ° C. for 2 hours.

  When the 3597th base of the 4th intron is normal (C / C), the molecular weights of the sections produced after enzymatic cleavage are 2209.4D (7mer) and 3988.6 (13mer) D (FIG. 7). When heterozygous (C / T), the molecular weights of the generated sections are 2209.4D, 2224.4D (more than 7mer), 3988.6D, and 3962.6D (13mer) (FIG. 8). On the other hand, when all were mutated to T (T / T), the molecular weights of the sections were 2224.4D (7mer) and 3962.6D (13mer) (FIG. 9).

Tyrosine-methionine-aspartate-aspartate (YMDD) site base mutation of hepatitis B virus DNA polymerase We investigated the base mutation of the YMDD site located in the DNA polymerase gene of the B virus-infected virus that induces hepatitis B in humans. . Resistance to lamivudine, a therapeutic agent for hepatitis B, is generated by base mutation at this site. It is known that resistance to lamivudine occurs when the methionine (M) of codon 552 is changed to valine (V) or isoleucine (I).

1. PCR amplification and restriction enzyme cleavage Hepatitis B virus DNA is extracted from 0.2 ml of serum using QIAamp blood kit (Qiagen, CA), 2 μl of which is used for PCR amplification.
The template DNA sequence (5 ′ → 3 ′) is as follows.
TTCCCCCACTGTTTGGCTTTCAGTTAT ATG GATGATTGGGTATTGGGGCCCAAGTCTGTA
( SEQ ID NO: 11)
The underlined sequence is the site where the following primers 5 and 6 bind.
Primer 5.5'-TTCCCCCACTGTTTGGCTggatgTCAGTTAT-3 '(31mer) (SEQ ID NO: 12)
Primer 6.5′-TACAGACTTGGCCCCACATACACCCAT G ATC-3 ′ (30mer) (SEQ ID NO: 13)
The sequence written in lower case in primer 5 is a sequence in which Fok I and BstF5I recognition sequences that are not present in Template DNA are artificially inserted. In primer 6, the underlined sequence is prevented from being recognized by Fok I. Therefore, it is an artificially modified array.
PCR using 20 mM TrisHCl (pH 8.4), 50 mM KCl, 0.2 mM dNTP, 0.4 U Platinum Taq Polymerase (Invitrogen, 10966-026), 18 μl of a reaction solution containing 10 pmol each of primers 5 and 6 under the following conditions Perform the reaction.
94 ° C for 2 minutes,
94 ° C 15 seconds 50 ° C 15 seconds 72 ° C 30 seconds (10 cycles)
94 ℃ 15 seconds 55 ℃ 15 seconds 72 ℃ 30 seconds (35cycles)

The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
TTCCCCCACTGTTTGGCTggatgTC AGTTATA TGGATCATGTGGTATTGGGGGCCAAGTCTGTA ( SEQ ID NO: 14)
AAGGGGGGTGACAAACCGAcctac AGTCAATATACACT AGTACACCATAACCCCCCGGTTCAGACAT ( SEQ ID NO: 15)
The site indicated in lower case is the sequence recognized by Fok I and BstF5I, and the underlined site is the sequence of the fragment produced by restriction enzyme cleavage. PCR reaction was mixed with 1U of FokI (NEB R109L), 1U of BstF5I (NEB, V0031L) and 10 μl of reaction solution (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT) at 37 ° C. for 2 hours, and The reaction is carried out at 45 ° C. for 2 hours. After first cutting with FokI for 2 hours at 37 ° C, BstF5I can be added to cut at 45 ° C for 2 hours.

2. Purification and desalting and MALDI-TOF mass spectrometry The same procedure as in Example 1 is performed.
The calculated size of the section produced by enzymatic cleavage and the value measured by actual molecular weight analysis agreed exactly to the extent that they showed a difference of 0.1% or less (Table 2).

In the above table, the resolution (the difference between the observed mass and the expected mass divided by the expected mass) is all 0.1% or less.

Base mutation at the 5 'NCR (Non Coding Region) site of hepatitis C virus When interferon is used for the treatment of chronic hepatitis C, it shows different therapeutic effects according to the genotype of human hepatitis C virus, before using interferon In addition, it is necessary to investigate the genotype of hepatitis C virus present in the body. In order to clarify the genotype, it is effective to investigate the 5′NCR base mutation, and this example describes a method for analyzing the base mutation at the 5′NCR site of hepatitis C virus.

1. RT PCR
Hepatitis C virus RNA is extracted from 0.14 ml of serum using QIAamp viral RNA Mini kit (Qiagen, CA), 10 μl of which is used for RT PCR amplification.
The reaction solution containing 0.2 mM dNTP, 0.4 μM primer 2, and 10 μl RNA is reacted at 65 ° C. for 5 minutes, and then left on ice for 1 minute. To this reaction solution, 20 mM TrisHCl (pH 8.4), 50 mM KCl, 4 mM DTT, 0.4 μM primer 1, 100 U SuperScript III RNase H-Reverse Transcriptase (Invitrogen, 18080-044), 20 U RNaseOUT (Invitrogen, 10777-019), RT PCR is performed under the following conditions using 25 μl of a reaction solution mixed with 0.4 U Platinum Taq Polymerase (Invitrogen, 10966-026).
50 ° C 45 minutes,
94 ° C for 2 minutes,
94 ℃ 15 seconds 55 ℃ 15 seconds 72 ℃ 15 seconds (35cycles)
72 ° C 5 minutes

The template DNA sequence (5 ′ → 3 ′) is as follows.
GCAGAAAGCGTCTAGCCATGGCGGT TAGTATGAGT (intermediate omission) ACTGCCTGATAGGGTGCTTGGCAG ( SEQ ID NO: 16)
The underlined sequence and the like are the sites where the following primers 7 and 8 bind.
Primer 7.5'-GCAGAAAGCGTCTAGCCATGGCGT-3 '(24mer) (SEQ ID NO: 17)
Primer 8.5′-CTCGCAAGCACCCTATCAGGGCAGT-3 ′ (24mer) (SEQ ID NO: 18)

2. Nested PCR and restriction enzyme cleavage The RT PCR reaction solution was diluted to 1/50, and 2 μl thereof was diluted with 20 mM TrisHCl (pH 8.4), 50 mM KCl, 0.2 mM dNTP, 0.4 U Platinum Taq Polymerase (Invitrogen, 10966-026). ) The following three kinds of PCR reactions and restriction enzyme treatments are performed using 18 μl of a reaction solution containing 10 pmol each of primers 9 and 10, 11 and 12, or 13 and 14. In reaction 1, primers 9 and 10 are used, in reaction 2 primers 11 and 12 are used, and in reaction 3 primers 13 and 14 are used. The PCR reaction temperature and time for all three reactions are as follows.
94 ° C for 5 minutes,
94 ℃ 30 seconds 55 ℃ 30 seconds 72 ℃ 30 seconds (35cycles)
72 ° C 5 minutes

1) Reaction <1>
PCR is performed on the RT-PCR reaction solution using primers 9 and 10. The template DNA sequence (5 ′ → 3 ′) is as follows.
CGTCTAGCCATGGCGTTTAGTATGAGGT CGTGCAGCCCTCCAGGACCCC ... (intermediate omitted)
... CTGCTAGCCGAGTAGTGTTGGGTCGCGAAAG GCCTTGTGGTACTGCCTGATAGGG ( SEQ ID NO: 19)
The underlined sequence is the site where the following primers 9 and 10 bind.
Primer 9.5'-CGTCTAGCCATGGCGGTTAGggatgATGAGTGT-3 '(32mer) (SEQ ID NO: 20)
Primer 10.5′-CCCTATCAGGGCAGTACCACAAGGC-3 ′ (24mer) (SEQ ID NO: 21)

The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
CGTCTAGCCATGGCGGTTAGggatgAT GAGTGTC GTGCAGCCTCCCAGGACCC ... (intermediate omitted)
GCAGATCGGTACCCCAATCCCTAC TACTCACAGCACCGTCGGAGGTCCTGGG ... (intermediate omitted)
CTGCTAGCCGAGTAGTGTTGGGTCGCGAAAAGCCCTGTGGTACTGCCTGATAGGG ( SEQ ID NO: 22)
... GACGATCGGCCTCATCACAACCCACGCGTTTCCGGAACACCATGACGGGACTATCCCC ( SEQ ID NO: 23)
The site indicated in lower case is the sequence recognized by Fok I and BstF5I, and the underlined site is the sequence of the fragment generated by restriction enzyme cleavage (7mer and 13mer). The PCR reaction was mixed with 1 U of FokI (NEB R109L), 1 U of BstF5I (NEB V0031L) and 10 μl of the reaction solution (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT) at 37 ° C. for 2 hours, and 45 The reaction is carried out at 2 ° C. for 2 hours. After first cutting with FokI for 2 hours at 37 ° C, BstF5I can be added to cut at 45 ° C for 2 hours.

2) Reaction <2>
PCR is performed on the RT-PCR reaction solution using primers 11 and 12. The template DNA sequence (5 ′ → 3 ′) is as follows.
GTGGTCTGCGGAACCGGTGAGTACACCCGGAAT TGCCAGGACGACCGGGTCC ... (intermediate omitted)
... CCCCCGCAAGACTC CTAGCCGAGTAGRGGTTGGGTRGCGAA ( SEQ ID NO: 24)
The underlined sequence and the like are the sites where the following primer sequences 11 and 12 bind.
Primer 11.5'-GTGGTCTGtccacCGGGTGAGTACACCCGGAAT-3 '(32mer) (SEQ ID NO: 25)
Primer 12.5′-TTCGCRACCCAACRCTCACTCCAACGGTCCCGCTAG-3 ′ (35mer) (SEQ ID NO: 26)
The base represented by R is adenine (A) or guanine (G), and a mixture of two kinds of primers containing each base is used.

The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
GTGGTCTGtccacCGGTGAGTACACCCGGAATTG CCAGGACGACCGG GTCC ... (intermediate omitted)
CACCAGACaggtgtGCCACTCATGTGCCCTTA ACGGTCCTGCTGGCCCAG G ... (intermediate omitted)
... CCCCGC AAGACTGCTAGCCG gaccgtttgaGTAGRGTTGGGTRGCGAA ( SEQ ID NO: 27)
... GGGG CGTTCTGACGATCGGCctg gcaacctCATCRCAACCCARCGCTT ( SEQ ID NO: 28)
The site written in lower case is the sequence recognized by Mme I and Ava II, and the underlined site is the sequence of the fragment generated by cleavage of the restriction enzyme (13mer, 18mer, 24mer, and 19mer). The PCR reaction product was mixed with 1.5 μM of MmeI (NEB R0637L), 50 μM SAM, and 1 × reaction solution (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9), and mixed at 37 ° C. for 2 hours. Then, 1.5 U of AvaII (NEB, R0153S) is added and the reaction is carried out at 37 ° C. for 2 hours. MmeI and AvaII can be added and reacted simultaneously.

3) Reaction <3>
PCR is performed on the RT-PCR reaction solution using primers 13 and 14. The template DNA sequence (5 ′ → 3 ′) is as follows.
GACIGGGTCCTTTCTTGGA TCAACCCGCTCAATGCCTGGAG ATTGGGCGGTGCCCCCGC ( SEQ ID NO: 29)
The underlined sequence is the site where the following primers 13 and 14 are bound,
Primer 13.5′-GACIGGGTCCCTggatgTCTGTG-3 ′ (23mer) (SEQ ID NO: 30)
Primer 14.5′-GCGGGGGCACggatgCCCAAAT-3 ′ (22mer) (SEQ ID NO: 31)
The base represented by I is inosine.
The sequence of the fragment generated via PCR is as follows (5 ′ → 3 ′).
GACIGGGTCCTggatagTC TTGGATCAAACCCGCTCAATGCCTGGAGATTTGGG catccGTGCCCCCGC ( SEQ ID NO: 32)
CTGICCCAGGAcctac AGAACCTAGTTGG GCGAGTTACGGACC TCTAAAC CCgtggCACGGGGGCG ( SEQ ID NO: 33)
The site indicated in lower case is the sequence recognized by Fok I and BstF5I, and the underlined site is the sequence of the fragment produced by restriction enzyme cleavage. There are two types of fragments , 2 types of 3mer, 2 types of 13mer, and 2 types of 14mer. PCR reaction was mixed with 1U of FokI (NEB R109L), 1U of BstF5I (NEB, V0031L) and 10 μl of reaction solution (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT) at 37 ° C. for 2 hours, and The reaction is carried out at 45 ° C. for 2 hours. After first cutting with FokI for 2 hours at 37 ° C, BstF5I can be added to cut at 45 ° C for 2 hours.

2. Purification, Desalting and MALDI-TOF Mass Spectrometry Three types of PCR and restriction enzyme cleavage reaction solutions are purified in the same manner as in Example 1 and the molecular weight is measured.
The sizes of the sections and the like generated by the reactions <1> (Table 3), <2> (Table 4), and <3> (Table 5) are as shown in the following Tables 3 to 5. The genotype of hepatitis C virus is determined based on the size of the section based on the quick reference table shown in Tables 3 to 5.

It is a 7-mer MALDI-TOF mass spectrometry diagram when the 2741st base of the 4th intron of the human maspin (serpinb5) gene is normal (C / C). It is a 13-mer MALDI-TOF mass spectrometry diagram when the 2741st base of the fourth intron of the human maspin gene is normal (C / C). It is a 7-mer MALDI-TOF mass spectrometry figure when the 2741st base of the 4th intron of a human maspin gene is hetero (C / T). It is a 13-mer MALDI-TOF mass spectrometric diagram when the 2741st base of the fourth intron of the human maspin gene is hetero (C / T). It is a 7-mer MALDI-TOF mass spectrometry diagram when the 2741st base of the fourth intron of the human maspin gene is all mutated to T (T / T). It is a 13-mer MALDI-TOF mass spectrometry diagram when the 2741st base of the fourth intron of the human maspin gene is all mutated to T (T / T). It is a 7-mer and 13-mer MALDI-TOF mass spectrometry diagram when the 3597th base of the fourth intron of the human maspin gene is normal (C / C). It is a 7mer and 13mer MALDI-TOF mass spectrometry figure when the 3597th base of the 4th intron of a human maspin gene is hetero (C / T). It is a 7mer and 13mer MALDI-TOF mass spectrometry figure when the 3597th base of the 4th intron of a human maspin gene is all mutated to T (T / T).

Claims (9)

a) PCR amplification of a specific polynucleotide fragment using a forward primer comprising a site recognized by two different type II restriction enzymes and a reverse primer comprising / excluding the restriction enzyme recognition site Gene amplification stage to
b) the amplified specific double-stranded polynucleotide was cut by the restriction enzyme, including single nucleotide polymorphism (SNP) site while having a size of 2 to 32 amino bases from the double-stranded polynucleotide fragment restriction enzyme cleavage step of generating two or more single-stranded polynucleotide fragments, and c) matrix-assisted laser desorption ionization - time of flight mass spectra (MALDI-TOF MS) 1 present of two or more of the above generated by analysis A base mutation analysis method comprising a molecular weight measurement step of measuring the molecular weight of each of the strand polynucleotide fragments . When 2 to 34 SNP sites are present in the specific double-stranded polynucleotide fragment, the SNP site is always present in any single-stranded polynucleotide fragment, but there is one specific SNP site. The base mutation analysis method according to claim 1, further comprising a restriction enzyme cleavage step of cleaving the specific double-stranded polynucleotide so as to be present only in the specific single-stranded polynucleotide fragment . a) Gene for PCR amplification of a specific polynucleotide fragment using a forward primer comprising a site recognized by two different types of restriction enzymes and a reverse primer comprising / not containing the restriction enzyme recognition site Amplification stage,
b) The amplified specific double-stranded polynucleotide is cleaved with the restriction enzyme so that a second restriction enzyme reacts while the first restriction enzyme reaction proceeds, and the double-stranded polynucleotide fragment is cleaved. A restriction enzyme treatment step for producing two or more kinds of single-stranded polynucleotide segments having a size of 2 to 32 bases and containing a SNP site, and c) the molecular weight of the produced single-stranded polynucleotide fragment is MALDI A base mutation analysis method including a molecular weight measurement step measured by TOF MS . The base mutation analysis method according to claim 1, wherein the forward primer is one primer selected from the group consisting of SEQ ID NOs: 2, 7, 12, 20, 25, and 30. . The base mutation analysis method according to any one of claims 1 to 3, wherein the restriction enzyme treatment step b) uses two types II restriction enzymes having optimum temperatures different from each other. The restriction enzyme FokI, BbvI, BsgI, BcgI, BpmI, BseRI and 1 and type of restriction enzymes optimum temperature selected from the group is in the range of 25 to 37 ° C. consisting BaeI, BstF5I, TaqI, BsaBI, BtrI, BstAPI, FauI, BclI, base mutation analysis of claim 5, optimum temperature selected from the group consisting of PciI and ApoI is characterized in that it is a one type II restriction enzymes which fall within the scope of 50-65 ° C. Way . The specific double-stranded polynucleotide fragment to be amplified in a) includes a tyrosine-methionine- aspartic acid - aspartic acid (YMDD) site which is an active site of hepatitis B virus DNA polymerase . The base mutation analysis method described in 1. The method for analyzing base mutation according to any one of claims 1 to 3, wherein the specific double-stranded polynucleotide fragment to be amplified in a) includes a part of the hepatitis C virus 5'-noncoding region (NCR) . The particular double-stranded polynucleotide fragment amplification: a) a base mutation analysis method according to claim 1 comprising a first 2741 or 3597 of the base moiety of the fourth intron of the human maspin gene.