HK1113589A1 - Method for detecting mutations - Google Patents

Abstract

A method for precisely and effectively detecting mutations of organism is provided. The method for detecting a mutation includes the steps of: a) amplifying a target polynucleotide using a forward primer and a reverse primer; b) generating fragments of two or more single-stranded polynucleotides including one or more mutations sequence having the size of 2-32 bases by cleaving the amplified polynucleotide with restriction enzymes; and c) measuring the molecular weight of the cleaved fragments.

Description

Method for detecting mutation

Technical Field

The present invention relates to a method for detecting genetic variation in an organism. Genetic analysis is used for risk assessment, diagnosis, prognosis or treatment of diseases. For example, mutation analysis of a specific gene for a specific person makes it possible to predict the risk of disease, thereby promoting prevention of disease. By mutation analysis, it is possible to detect whether or not a virus causing a disease has developed resistance to a drug, and thus effective treatment can be performed.

Background

The human genome project enables us to more fully measure the risk, diagnosis or prognosis of a disease and predict the response to a drug treatment. Analysis of the nucleotide sequence of a large number of individuals exhibits polymorphic sites, which are called SNPs (single nucleotide polymorphisms). SNPs are variations that occur at a specific frequency in the nucleotide sequence of a chromosome of an organism. In humans, SNPs occur approximately every 1,000 bases. Considering the size of the human genome, millions of SNPs exist in the human body. Since SNPs are regarded as a means of explaining characteristic differences between individuals, they can be used for prevention or treatment of diseases by diagnosing causes.

SNPs discovered by the human genome project only indicate that polymorphisms are present in humans, but do not indicate how those polymorphisms are associated with disease. In order to reveal the relationship between SNPs and diseases, it is necessary to comparatively analyze polymorphism patterns manifested in healthy persons and patients, i.e., SNP scores. In order to accurately examine the relationship between SNPs and diseases, a large number of SNPs should be correctly analyzed.

SNP scoring methods include DNA sequencing, PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism), allele-specific hybridization, oligonucleotide ligation (oligo-ligation), micro-sequencing (mini-sequencing), and enzymatic cleavage. A method using a DNA chip has also been proposed, but this method is not different from allele-specific hybridization in principle except for using a carrier to which an oligonucleotide probe is immobilized.

Two traditional methods for performing DNA sequencing are the Maxam & Gilbert chemical sequencing method, and the recently used Sanger method. DNA sequencing is intended to clarify the nucleotide sequence of the whole or part of a gene, rather than to detect genetic variations at specific sites. Since genetic variation at a specific site can be recognized by detecting a nucleotide sequence, a DNA sequencing method can be used in SNP scoring. However, DNA sequencing is not very efficient because adjacent nucleotide sequences that do not need to be detected can be read out using the target SNP.

In PCR-SSCP (ORITA, M.et. al., Genomics, 1989, 5: 8874-8879), the SNP-containing sequence to be analyzed is amplified by PCR and then divided into single strands. Thereafter, electrophoresis was performed on polyacrylamide gel. Since the difference in sequence changes the secondary structure of DNA, mutations in the sequence can be detected by the difference in electrophoretic migration velocity due to the difference in structure.

Allele-specific hybridization is the detection of mutations by modulating hybridization conditions, such as temperature, using radioisotope-labeled DNA to hybridize with probes attached to nylon membranes.

Oligonucleotide ligation (Nucleic Acid Research 24, 3728, 1996) is to detect sequence mutations by performing a reaction under conditions in which the reaction does not occur if the target DNA is not complementary to the template DNA, thereby confirming whether ligation occurs.

Micro sequencing (Genome Research 7: 606, 1997) was developed for SNP scoring. This method performs DNA polymerization under conditions where only one target base can be polymerized, and then identifies what the polymerized base is.

These methods are not very efficient methods for analyzing many samples, since PCR-SSCP, allele-specific hybridization, and oligonucleotide ligation all use polyacrylamide gels. Also, these methods are not able to recognize errors caused by probe mismatches to non-target sites.

Although micro-sequencing is simple and effective in analyzing many samples, incorrect results due to errors in mismatch cannot be recognized, and deletion and insertion of bases cannot be found by micro-sequencing.

For SNP scoring, enzymatic cleavage was also developed (WO 01/90419). In the enzymatic cleavage method, a sequence to be analyzed is amplified using an appropriate method such as PCR. The amplification product includes a sequence that can be cleaved or recognized by two restriction enzymes. The enzymatic cleavage method is to detect sequence variation by cleaving the amplified product with two restriction enzymes and detecting the molecular weight of the cleaved fragments. Since the molecular weight of the fragment obtained by the restriction enzyme reaction can be detected by mass spectrometry after the gene is amplified by PCR, the enzymatic cleavage method has the advantages of simplicity and rapidness. However, the enzymatic cleavage described in patent WO 01/90419 does not allow to identify an incorrect analysis caused by errors. Although incorrect analysis may result when primers bind to non-target sites during PCR, the incorrect analysis cannot be identified. For example, a primer for detecting a polymorphism of CYP2C9 may be bound to CYP2C 8. In this case, it is difficult to find whether an error has occurred because it is not possible to identify whether the primer binds to CYP2C8 in addition to CYP2C 9. This method can detect substitution of one base, but cannot detect deletion or insertion of a base. Furthermore, this method cannot detect substitution of two or more adjacent bases at the same time.

Disclosure of Invention

Accordingly, an object of the present invention is to provide a method for detecting a mutation in an organism accurately and efficiently.

In order to achieve the above object, in an embodiment of the present invention, a method for accurately and efficiently detecting a mutation in an organism is provided.

The present invention can simply and rapidly detect mutations in many samples, and can accurately detect mutations by recognizing errors caused by the binding of primers in the wrong regions. Furthermore, the present invention provides a method for detecting two or more mutation sites which are adjacent to each other at the same time within 32 bases; the invention also provides methods for detecting deletions or insertions. In particular, when there are different genotypes in an individual, the method of the present invention can identify whether mutations at different sites exist simultaneously in one genotype or are present in a mixture in different genotypes. For example, a human has a pair (two) of chromosomes that contain the same genetic information. When a mutation occurs, it may occur either in two chromosomes (homozygote) or in one chromosome (heterozygote). When mutations of two or more adjacent bases are all heterozygous, those mutations may be present in one chromosome at the same time, or may be present in different chromosomes. Since these two situations may have different effects on life, they should be distinguished. In the case of human infection with viruses, various genotypes are mixed. When mutations of two or more adjacent bases are all heterozygous, it should be distinguished whether those mutations are present in one genotype at the same time or in different genotypes.

In order to analyze a mutation, the method of the present invention amplifies a target sequence to include a site of a resultant product that can be cut by a restriction enzyme, and controls the number of bases in a fragment cut by the restriction enzyme to be less than 32, and at least one base among them is generated by replication of a template other than a primer itself, and after the amplified fragment is cut by the restriction enzyme, the molecular weight of the fragment is measured to analyze the mutation.

In one embodiment of the present invention, there is provided a method for detecting a mutation, comprising:

a) amplifying the target polynucleotide using a forward primer and a reverse primer;

b) digesting the amplified target polynucleotide with restriction enzymes to generate two or more single-stranded polynucleotide fragments comprising one or more mutant sequences having a length of 2 to 32 bases; and

c) the molecular weight of the cleaved fragment (cleaved fragment) was determined.

Preferably, the amplified polynucleotide is cleaved to contain one of two or more different mutations in one single-stranded polynucleotide fragment and all mutations in another single-stranded nucleotide fragment. For example, when a and G in the.

In order to analyze the mutation, the method of the present invention amplifies the target sequence so as to include sites at which the amplified product can be cleaved with restriction enzymes, and the cleaved fragments have the following structures.

5’-

Primer binding sequence 1

Recognition sequence of restriction enzyme

Primer binding sequence 2

Pre-mutation sequence

Mutant sequences

Post-mutation sequence

Primer binding sequence 3

-3’

The "restriction enzyme recognition sequence" refers to a sequence that can be recognized simultaneously or adjacently by different restriction enzymes, and the sequence may not correspond to the cleavage sequence. For example, both FokI and BSTFSI recognize the sequence GGATG. However, the cleavage sites are adjacent to the 9 th/13 th and 2 nd/0 th bases from the 3' end of the recognition sequence, respectively. The two restriction enzymes used to recognize the restriction enzyme recognition sequences may have the same or different optimal temperatures. It is preferable to have different optimum temperatures. Preferably, the restriction enzymes are restriction enzymes with lower optimal temperature selected from FokI, Bbv I, Bsg I, Bcg I, BpmI, BseR I, Bae I and restriction enzymes with higher optimal temperature selected from BSTF5I, Taq I, BsaB I, Btr I, BstAP I, Fau I, Bcl I, Pci I, Apo 1. More preferably, the restriction enzymes are Fok 1 and BSTF 5I.

The restriction enzymes with lower optimal temperature include Bae I (25 ℃), FokI, BbvI, Bsg I, Bcg I, Bpm I, BseR I, Mmel I, Ava II (37 ℃); the restriction enzymes with higher temperature optima were BSTF5I, Taq I (65 ℃), BsaB I, Btr I, BstAP I (60 ℃), Bcl I, Pci I, Apo I (50 ℃).

One of the two primers used for PCR amplification comprises primer binding sequence 1, restriction enzyme recognition sequence and primer binding sequence 2, while the other primer comprises primer binding sequence 3.

The "primer binding sequence" is a sequence complementary to a nucleic acid as a template, but the restriction enzyme recognition sequence may not be complementary to the nucleic acid. The number of bases of the primer binding sequences 1, 2 and 3 should be at least four or more to bind to the template DNA. Since the primer can be well combined with the template DNA at a length of 8 to 30 bases, the number of bases is preferably 8 to 30. The "pre-mutation sequence" is the sequence towards the 5' end of the mutation to be detected. The "mutation sequence" is the sequence corresponding to the mutation to be detected. Substitution, insertion and deletion of bases may occur, wherein the number of bases is usually 1, and may be two or more. The "post-mutation sequence" (sequence after mutation) is a sequence toward the 3' end of the mutated sequence.

Preferably, the total number of bases of the pre-mutation sequence and the post-mutation sequence is one or more. The fragment cut by the restriction enzyme should include a mutant sequence, and the length of the fragment is preferably 2 to 32 bases. More preferably, it is 12 bases in length. The reason for limiting the fragment length is that mass spectrometry results better with fragments of the preferred length. The number of bases of the above-mentioned fragment is preferable because the fragment having a length exceeding 32 bases is too long to detect a mutation by measuring the molecular weight using mass spectrometry. Furthermore, since a fragment having only one mutated sequence cannot recognize the binding of a primer at an erroneous site, a fragment having only one base is not preferable. Since the two restriction enzymes recognize the same or adjacent sites, it is preferable that when one restriction enzyme reacts with the amplification product, the other restriction enzyme is inactive. When the amplified fragment is digested with restriction enzymes, the reaction can be continuously performed at different temperatures in consideration of the optimal temperatures of the two restriction enzymes. Alternatively, the fragment may be cut with one restriction enzyme and then with another restriction enzyme. Here, cleavage with the first restriction enzyme should not eliminate or destroy the recognition sequence or cleavage site of the second restriction enzyme present in the fragment comprising the mutated sequence.

Brief description of the drawings

The above and other features, aspects, and advantages of preferred embodiments of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

FIG. 1 is a MALDI-TOF mass spectrum (C/C) of 7mer when base 2741 of intron 4 of human maspin (serpinb5) gene is normal;

FIG. 2 is a MALDI-TOF mass spectrum (C/C) of a 13mer when base 2741 of intron 4 of the human maspin gene is normal;

FIG. 3 is a 7mer MALDI-TOF mass spectrum (C/T) when base 2741 of intron 4 of human maspin gene is heterozygous;

FIG. 4 is a MALDI-TOF mass spectrum (C/T) of 13mer when base 2741 of intron 4 of human maspin gene is heterozygous;

FIG. 5 is a MALDI-TOF mass spectrum (T/T) of 7mer when all of bases at position 2741 of intron 4 of the human maspin gene are changed to T;

FIG. 6 is a MALDI-TOF mass spectrum (T/T) of 13mer when all of bases at position 2741 of intron 4 of the human maspin gene are changed to T;

FIG. 7 is a MALDI-TOF mass spectrum (C/C) of 7mer and 13mer when the 3597 th base of the 4 th intron of the human maspin gene is normal;

FIG. 8 is a mass spectrum (C/T) of MALDI-TOF of 7mer and 13mer when the 3597 th base of the 4 th intron of the human maspin gene is a hybrid; and

FIG. 9 shows MALDI-TOF mass spectra (T/T) of 7mer and 13mer when all of the 3597 th bases of the 4 th intron of the human maspin gene were changed to T.

Best mode for carrying out the invention

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1 mutation of 2741 st base in the 4 th intron of human maspin Gene

A mutation at base 2741 (rs 1509477, base 61001755 of chromosome 18) of the human maspin (serpinb5) gene known as a cancer metastasis suppressor gene was detected.

1. PCR amplification and restriction enzyme digestion

The sequence of the template DNA (5 '→ 3') is as follows:

GTTTCACTTGATAAAGCAATAAAATGCTATTCAcAGCTGCATGAGGC TACACCCTTCTTTTGAATGCAG(SEQ ID NO：1)

underlined sequences are sites that hybridize to the following primers 1 and 2. The base indicated by lower case letters is a "mutated sequence".

Primer 1: 5'-TCACTTGATAAAGCAATAAAAggatgGCTATTCA-3' (34mer) (SEQ ID NO: 2)

Primer 2: 5'-CATTCAAAAGAAGGGTGTAGCCTCATGC-3' (28mer) (SEQ ID NO: 3)

The sequences indicated in lower case letters are the identification sequences of FokI and BSTF 5I.

PCR buffer (1X), 2mM MgSO₄200mM deoxynucleotide triphosphate (dNTP), 0.315U platinumTaq polymerase (Invitrogen, 10966-026), 0.5. mu.M primer 1 and 0.5. mu.M primer 2, and 36ng genomic DNA to a total reaction volume of 18. mu.l. Then, PCR reaction was performed under the following conditions.

The temperature of the mixture is 94 ℃ for 2 minutes,

94 ℃, 15 seconds 55 ℃, 15 seconds 72 ℃, 30 seconds (10 cycles),

94 ℃, 15 seconds 60 ℃, 15 seconds 72 ℃, 30 seconds (35 cycles)

Genomic DNA was isolated from blood and purified. For example, the "SDS/proteinase K" method (Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) or the QIAamp DNA minikit 250(Qiagen 51106) can be used for isolating DNA from blood. When the concentration of DNA is low, the DNA can be concentrated by the following method. 1/10 volumes of 3M sodium acetate (pH 5.3) and 2.5 volumes of ethanol were first added to the DNA solution, slowly mixing. The resulting solution was left at-20 ℃ for more than 1 hour and then centrifuged at 13000 rpm for 15 minutes at 4 ℃. After removing the supernatant, 70% ethanol was added, and the resulting solution was centrifuged at 13000 rpm for 10 minutes at 4 ℃. Then, ethanol was dried off, and a predetermined volume of distilled water was added to the resulting solution.

The sequence of the fragment obtained by PCR is shown below (5 '→ 3').

TCACTTGATAAAGCAATAAAAggatgGCTATTCA[C/T]AGCTGCATGAGGCTACACCCTTCTTTTGAATG(SEQ ID NO：4)

AGTGAACTATTTCGTTATTTTcctacCGATAAGT[G/A]TCGACGTACTCCGATGTGGGAAGAAAACTTAC(SEQ ID NO：5)

The sites indicated by lower case letters are the sequences recognized by FokI and BstF5I, the underlined sites are the sequences of the fragments generated by restriction enzyme digestion, and the bases indicated by brackets are the "mutated sequences". To the reaction was added 1U FokI (NEB R109L), 1U BstF5I (NEB, V0031L), 50mM potassium acetate, 20mM Tris (hydroxymethyl) aminomethane acetate (Tris-acetate), 10mM magnesium acetate, 1mM Dithiothreitol (DTT) (pH7.925 ℃ C.). The resulting solution was reacted at 25 ℃ for 2 hours followed by 45 ℃ for 2 hours.

To optimize the enzymatic reactions, FokI and BstF5I were reacted with the amplification products at 25 ℃, 37 ℃, 45 ℃, 55 ℃ and 65 ℃. As a result, in the presence of FokI, 70% of the enzymatic reactions were carried out at 25 ℃ and more than 90% of the enzymatic reactions were carried out at 37 ℃. In the presence of BstFSI, the enzyme reaction did not occur at 25 ℃. Therefore, it is preferable that the amplification product is first reacted at 25 ℃ where only FokI can be reacted, and then, reacted at more than 37 ℃ where BstF5I can be reacted.

2. Purification and desalination

Preferably, the DNA fragment is purified and isolated from the above solution treated with the restriction enzyme, and then the molecular weight of the fragment is measured. For example, a nucleic Genotyping kit (variaginics, USA) may be used. Mu.l of 1M TEAA (triethylammonium acetate, pH 7.6) was added to the restriction enzyme reaction solution and allowed to stand for 1 minute. 70. mu.l of 1M TEAA and 90. mu.l of the above mixed solution were added to a Sample preparation plate (Sample preparation plate), and then the Sample preparation plate was filtered five times with 85. mu.l of 1M TEAA. The sample preparation plate was centrifuged at 1000 rpm for 5 minutes. Thereafter, the sample preparation plate was placed on a collection plate (collection plate), and 60 μ l of 60% isopropyl alcohol was added thereto and filtered. When the effluent was collected in a collection plate, the plate was dried at 115 ℃ for 75 minutes.

3. MALDI-TOF mass spectrometry

Mu.l of MALDI matrix (22.8mg sodium citrate, 148.5mg hydroxypicolinic acid, 1.12ml acetonitrile, 7.8ml water) was added to the collection plate, and then 4. mu.l of the mixture of MALDI matrix and effluent was placed on the base plate of MALDI-TOF chip (Anchor chip plate, Biflex IV, Bruker). Dried at 37 ℃ for 30 minutes, left to cool at room temperature for a while, and then subjected to MALDI-TOF analysis. The analytical method was referred to the MALDI-TOF manual.

When the 2741 st base of the 4 th intron is normal (C/C), the fragments obtained after cleavage have molecular weights of 2135.4D (daltons) (7mer) and 4078.6D (13mer) (see FIGS. 1 and 2). When the 2741 st base of the 4 th intron is heterozygous (C/T), the molecular weights of the fragments are 2135.4D, 2150.4D (7mer) and 4078.6D, 4062.6D (13mer) (see FIGS. 3 and 4). When the 2741 st base of the 4 th intron is changed to T (T/T), the molecular weight of the fragment is 2150.4D (7MER) and 4062.6D (13MER) (see FIGS. 5 and 6).

Example 2 mutation of 3597 th base of the fourth intron of the human maspin (serpinb5) (rs 1396782; 61002611 th base of human chromosome 18) gene, which is known as a human cancer metastasis suppressor gene

The sequence of the template DNA is shown below.

CTGGAGTATTATCCTTGCAGGCTTGATATGAAGcTTGAAATTTCTCC CCAAAGAGATTTAGTTAACAGGCAAA(SEQ ID NO：6)

Underlined sequences are sites that hybridize to primers 3 and 4 described below. Mutations in lower case letters are "mutated sequences".

Primer 3: 5'-GAGTATTATCCTTGCAGGCTTggatgATATGAAG-3' (34mer) (SEQ ID NO: 7)

Primer 4: 5'-GCCTGTTAACTAAATCTCTTTGGGGAGAA-3' (29mer) (SEQ ID NO: 8)

In the above primer, the sites indicated by lower case letters are sequences which are not present in the template DNA, but FokI and BstF5I can recognize them. The experimental procedure involving the PCR reaction was the same as described in example 1.

The sequence of the fragment obtained by PCR is shown below (5 '→ 3').

GAGTATTATC CTTGCAGGCTTggatgATATGAAG[C/T]TTGAAATTTCTCCCCAAAGAGATTTAGTTAACAGGC(SEQ ID NO：9)

CTCATAATAGGAACGTCCGAAcctacTATACTTC[G/A]AACTTTAAAGAGGGGTTTCTCTAAATCAATTGTCCG(SEQ ID NO：10)

In the above sequences, the sites indicated by lower case letters are recognition sequences of restriction enzymes, the underlined sites are sequences of fragments obtained by restriction enzyme digestion, and the bases indicated by brackets ([ ]) are "mutated sequences". 1U FokI (NEB R109L), 1U BstF5I (NEB, V0031L), 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 1mM DTT (pH7.925 ℃ C.). The resulting solution was reacted at 25 ℃ for 2 hours followed by 45 ℃ for 2 hours.

When the 3597 th base of the 4 th intron was normal (C/C), the fragments obtained by the cleavage were 2209.4D (7MER) and 3988.6D (13MER) (see FIG. 7). When the 3597 th base of the 4 th intron was heterozygous (C/T), the molecular weights of the fragments were 2209.4D, 2224.4D (7mer), and 3988.6D, 3972.6D (13mer) (see FIG. 8). When all of the 3597 th bases of the 4 th intron were changed to T (T/T), the molecular weights of the fragments were 2224.4D (7mer) and 3972.6D (13mer) (see FIG. 9).

Example 3 base mutation of tyrosine-methionine-aspartic acid (YMDD) site of hepatitis B Virus DNA polymerase

Detecting a mutation of YMDD site, which is located in DNA polymerase of hepatitis B virus causing human hepatitis B. Mutations in the YMDD site can confer resistance to lamivudine (lamivudine) for the treatment of hepatitis b. It is known that when methionine (M), i.e., codon 552, is changed to valine (V) or isoleucine (I), resistance to lamivudine is developed.

1. PCR amplification and restriction enzyme digestion

Hepatitis B virus DNA was isolated from 0.2ml serum using QIAamp blood kit (Qiagen, CA), and 2. mu.l of the DNA was used for PCR amplification.

The sequence of the template DNA (5 '→ 3') is shown below.

TTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGG GGGCCAAGTCTGTA (SEQ ID NO：11)

Underlined sequences are sites that hybridize to the following primers 5 and 6.

Primer 5(SEQ ID NO: 12):

5′-TTCCCCCACTGTTTGGCTggatgTCAGTTAT-3′(31mer)

primer 6(SEQ ID NO: 13):

5′-TACAGACTTGGCCCCCAATACCACATGATC-3′(30mer)

the sequences indicated in primer 5 by lower case letters are the recognition sequences of FokI and BstF5I, which are not contained in the template DNA, but are inserted artificially. The underlined sequence in primer 6 is an artificially altered sequence to prevent identification of fokl.

A PCR reaction was carried out under the following conditions by using 18. mu.l of a reaction solution containing 20mM Tris-HCl buffer (Tris-HCl, pH 8.4), 50mM potassium chloride, 0.2mM dNTP, 0.4U platinumTaq polymerase (Invitrogen, 10966-026), 10pmol of primer 5 and 10pmol of primer 6.

94 ℃ for 2 minutes

94 ℃, 15 seconds 50 ℃, 15 seconds 72 ℃, 30 seconds (10 cycles),

94 ℃, 15 sec 55 ℃, 15 sec 72 ℃, 30 sec (35 cycles)

The sequence (5 '→ 3') of the fragment obtained by PCR is shown below.

TTCCCCCACTGTTTGGCTggatgTCAGTTATATGGATCATGTGGTATTGGGGGCCAAGTCTGTA(SEQ ID NO：14)

AAGGGGGTGACAAACCGAcctacAGTCAATATACCTAGTACACCATAACCCCCGGTTCAGACAT(SEQ ID NO：15)

The sites indicated in lower case letters are sequences recognizable by FokI and BstF5I, and the underlined sites are sequences of fragments cut by restriction enzymes. The PCR product was mixed with 1U FokI (NEBR109L), 1U BstF5I (NEB, V0031L) and 10. mu.l of reaction solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 1mM DTT). The mixed solution was reacted at 37 ℃ for 2 hours and then at 45 ℃ for 2 hours. First, the PCR product was digested with FokI at 37 ℃ for 2 hours, and then with BstF5I at 45 ℃ for 2 hours.

2. Purification and desalting, and MALDI-TOF mass spectrometry

The experiment was carried out in the same manner as in example 1.

The theoretical length of the fragments obtained by cleavage corresponds exactly to the value determined by the actual molecular weight analysis, with a difference of less than 0.1% (see Table 1).

TABLE 1

Presumption and observation of the quality of oligonucleotides obtained by restriction endonuclease cleavage of PCR products

Predicting the sequence of fragments

Estimation of fragment Mass (Da)

Observation of fragment Mass (Da)

Genotype codon No.552

7mer 13mer

7mer 13mer

7mer 13mer

YMDD aTgYVDD aTgYIDD aTtYIDD aTcYIDD aTa

AGTTATa TCcAtATAACTGAAGTTATg TCcAcATAACTGAAGTTATa TCaAtATAACTGAAGTTATa TCgAtATAACTGAAGTTATa TCtAtATAACTGA

2199.4 3997.62215.4 3982.62199.4 4021.62199.4 4037.62199.4 4012.6

2199.6 3998.02215.9 3982.62199.6 4021.82199.6 4038.02199.6 4012.6

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

Example 4 mutation of 5' NCR (non-coding region) site of Hepatitis C Virus (HCV)

When interferon is used for the treatment of chronic hepatitis c, the difference in therapeutic effect expressed depends on the genotype of hepatitis c in a human body. Therefore, the genotype of hepatitis C virus in human should be examined before interferon is used. In order to find the genotype, detection of 5' NCR mutations is useful. In one embodiment of the present invention, a method for analyzing 5' NCR site mutations of hepatitis C virus is disclosed.

1. Reverse transcription polymerase chain reaction (RT PCR)

Hepatitis C virus RNA was isolated from 0.14ml serum using a QIAamp viral RNA minikit (Qiagen, CA) and 10. mu.l of RNA was used for RT PCR amplification.

The reaction solution containing 0.2mM dNTP, 0.4. mu.M primer 2 and 10. mu.l RNA was reacted at 65 ℃ for 5 minutes and then left on ice for 1 minute. The reaction solution was mixed with 20mM Tris HCl (pH 8.4), 50mM KCl, 4mM DTT, 0.4. mu.M primer 1, 100U SuperScript III ribonuclease H-reverse transcriptase (Invitrogen, 18080-. Then, RT PCR was performed using 25. mu.l of the obtained solution under the following conditions.

The temperature of the mixture is 50 ℃ for 45 minutes,

the temperature of the mixture is 94 ℃, the time of the mixture is 2 seconds,

94 ℃, 15 sec 55 ℃, 15 sec 72 ℃, 30 sec (35 cycles)

72 ℃ for 5 minutes

The sequence of the template DNA (5 '→ 3') is shown below.

GCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGT (omitted)ACTGCCTGATAGGGTGCTTGCGAG(SEQ ID NO：16)

The underlined sequences are the sites that hybridized to primers 7 and 8.

Primer 7(SEQ ID NO: 17): 5'-GCAGAAAGCGTCTAGCCATGGCGT-3' (24mer)

Primer 8(SEQ ID NO: 18): 5'-CTCGCAAGCACCCTATCAGGCAGT-3' (24mer)

2. Nested PCR (nested PCR) and restriction enzyme digestion

The RT PCR reaction was diluted to 1/50. Mu.l of the dilution was mixed with 18. mu.l of a reaction solution containing 20mM Tris HCl (pH 8.4), 50mM KCl, 0.2mM dNTP, 0.4U platinumTaq polymerase (Invitrogen, 10966-026), 10pmol of primers 9 and 10, 10pmol of primers 11 and 12, and 10pmol of primers 13 and 14. The following three types of PCR reactions and restriction enzyme treatments were carried out using the mixed solution. Primers 9 and 10 were used for reaction 1, primers 11 and 12 were used for reaction 2, and primers 13 and 14 were used for reaction 3. The PCR reaction temperature and time for the three reactions are as follows.

The temperature of the mixture is 94 ℃ for 5 minutes,

94 ℃, 30 seconds 55 ℃, 30 seconds 72 ℃, 30 seconds (35 cycles)

72 ℃ for 5 minutes

1) Reaction 1

This PCR amplification was performed in RT-PCR solution using primers 9 and 10. The sequence of the template DNA (5 '→ 3') is shown below.

CGTCTAGCCATGGCGTTAGTATGAGTGTCTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGG(SEQ ID NO：19)

Underlined sequences are sites that hybridize to the following primers 9 and 10.

Primer 9(SEQ ID NO: 20):

5′-CGTCTAGCCATGGCGTTAGggatgATGAGTGT-3′(32mer)

primer 10(SEQ ID NO: 21):

5′-CCCTATCAGGCAGTACCACAAGGC-3′(24mer)

the sequence of the fragment resulting from the PCR amplification is shown below (5 '→ 3').

CGTCTAGCCATGGCGTTAGggatgATGAGTGTCGtgcagcctccaggaccc. (omitted)

GCAGATCGGTACCGCAATCcctacTACTCACAGCACGTCGGAGGTCCTGGG. (omitted)

...CTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGG(SEQ ID NO：22)

...GACGATCGGCTCATCACAACCCAGCGCTTTCCGGAACACCATGACGGACTATCCC(SEQ ID NO：23)

The positions indicated in lower case letters are sequences recognizable by FokI and BstF 5I. The underlined sites are the sequences of the fragments generated by the restriction enzymes (7mer and 13 mer). The PCR product was mixed with 1U FokI (NEB R109L), 1U BstF5I (NEB, V0031L) and 10. mu.l of reaction solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 1mM DTT). The mixed solution was reacted at 37 ℃ for 2 hours and then at 45 ℃ for 2 hours. The PCR product was digested with FokI at 37 ℃ for 2 hours and then BstF5I at 45 ℃ for 2 hours.

2) Reaction 2

This PCR amplification was performed in RT-PCR solution using primers 11 and 12. The sequence of the template DNA (5 '→ 3') is shown below.

GTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGTCC

...CCCGCAAGACTGCTAGCCGAGTAGRGTTGGGTRGCGAA(SEQID NO：24)

Underlined sequences are sites hybridizing to the following primers 11 and 12.

Primer 11(SEQ ID NO: 25):

5′-GTGGTCTGtccaacCGGTGAGTACACCGGAAT-3′(32mer)

primer 12(SEQ ID NO: 26):

5′-TTCGCRACCCAACRCTACtccaacggtcCGGCTAG-3′(35MER)

the base represented by R is adenine (A) or guanine (G). A mixture of two primers containing each base was used.

The sequence of the fragment produced by this PCR is shown below (5 '→ 3').

GTGGTCTGtccaacCGGTGAGTACACCGGAATTGCCAGGACGACCGGGtcc

CACCAGACaggttgGCCACTCATGTGGCCTTAACGGTCCTGCTGGCCC AG(omit)

...CCCCGCAAGACTGCTAGCCGgaccgttggaGTAGRGTTGGGTRGCGAA(SEQ ID NO：27)

...GGGGCGTTCTGACGATCGGCctggcaacctCATCRCAACCCARCGCTT(SEQ ID NO：28)

The sites indicated in lower case letters are the sequences recognizable by MmeI and AvaII, and the underlined sites are the sequences of the fragments generated by restriction enzyme digestion (13mer, 18mer, 24mer, 19 mer). The PCR product was mixed with 1.5U MmeI (NEB R0637L), 50. mu.M SAM (S-adenosyl-L-methionine) and 10. mu.l of 1 × reaction solution (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 1mM DTT, pH 7.9). The mixture was reacted at 37 ℃ for 2 hours, and then 1.5U of AvaII (NEB, R0153S) was added thereto. The resulting solution was reacted at 37 ℃ for 2 hours. MmeI and AvaII may be added to the mixture simultaneously.

3) Reaction 3

This PCR amplification was performed in RT-PCR solution using primers 13 and 14. The sequence of the template DNA (5 '→ 3') is shown below.

GACIGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTG GGCGTGCCCCCGC(SEQ ID NO：29)

Underlined sequences are sites that hybridize to the following primers 13 and 14.

Primer 13(SEQ ID NO: 30):

5′-GACIGGGTCCTggatgTCTTGGA-3′(23mer)

primer 14(SEQ ID NO: 31):

5′-GCGGGGGCACggatgCCCAAAT-3′(22mer)

the base represented by I is inosine.

The sequence of the fragment produced by this PCR is shown below (5 '→ 3').

GACIGGGTCCTggatgTCTTGGATC AACCCGCTCAATGC CTGGAGATTTGGG catccGTGCCCCCGC(SEQ ID NO：32)

CTGICCCAGGAcctacAGAACCTAGTTGG GCGAGTTACGGACC TCTA AACCCgtaggCACGGGGGCG(SEQ ID NO：33)

The sites indicated in lower case letters are the sequences recognizable by FokI and BstF5I, and the underlined sites are the sequences of the fragments generated by restriction enzyme digestion. The fragments generated were 27 mers, 213 mers, and 2 14 mers. The PCR product was mixed with 1U FokI (NEB R109L), 1U BstF5I (NEB, V0031L) and 10. mu.1 reaction (50mM potassium acetate, 20mM Tris-acetate, 10mM potassium acetate, 1mM DTT). The mixture was reacted at 37 ℃ for 2 hours and then at 45 ℃ for 2 hours. The fragment was first digested with FokI at 37 ℃ for 2 hours and then with BstF5I at 45 ℃ for 2 hours.

2. Purification and desalting, and MALDI-TOF mass spectrometry

Three types of PCR and restriction enzyme reaction solutions were purified by the same method as in example 1, and then the molecular weights were measured.

The lengths of the fragments produced by reaction 1 (Table 2), reaction 2 (Table 3) and reaction 3 (Table 4) are shown in tables 2 to 4. The genotype of hepatitis C was determined from the fragment lengths obtained from the simple calculation table, as shown in tables 2 to 4.

TABLE 2

Genotype(s)	7mer	13mer
			1a	2216.4	3983.6
1b	2216.4	3983.6
			1c	2216.4	3983.6
3a	2216.4	3983.6
			3b	2216.4	3983.6
3c	2216.4	3983.6

3d	2216.4	3983.6
			3e	2216.4	3983.6
3f	2216.4	3983.6
			6b	2216.4	3983.6
7a	2216.4	3983.6
			7b	2216.4	3983.6
7c	2216.4	3983.6
			2′	2216.4	3989.6
5a	2216.4	3989.6
			1b	2216.4	3998.6
1d	2216.4	3998.6
			1e	2216.4	3998.6
1f	2216.4	3998.6
			2a	2216.4	3998.6
2b	2216.4	3998.6
			2c	2216.4	3998.6
2d	2216.4	3998.6
			2e	2216.4	3998.6
2′	2216.4	3998.6
			4h	2216.4	3998.6
6a	2216.4	3998.6
			7d	2216.4	3998.6
1b	2231.4	3967.6
			4g	2231.4	3967.6
4k	2231.4	3967.6
			2a	2231.4	3982.6
4a	2231.4	3982.6
			4b	2231.4	3982.6
4c	2231.4	3982.6
			4d	2231.4	3982.6

4e	2231.4	3982.6
			4e′	2231.4	3982.6
4f	2231.4	3982.6
			4f’	2231.4	3982.6

TABLE 3

Genotype(s)	13mer	18mer	Genotype(s)	14mer	19mer
						1a	4049.6	5556.6	2a	4337.8	5891.8
1b	4049.6	5556.6	2e	4337.8	5891.8
						1c	4049.6	5556.6	4b	4337.8	5891.8
1d	4049.6	5556.6	4e′	4337.8	5891.8
						1e	4049.6	5556.6	1a	4352.8	5875.8
1f	4049.6	5556.6	1c	4352.8	5875.8
						6b	4049.6	5556.6	1d	4352.8	5875.8
7a	4049.6	5556.6	1e	4352.8	5875.8
						7b	4049.6	5556.6	2a	4352.8	5875.8
4a	4049.6	5572.6	2b	4352.8	5875.8
						6a	4064.6	5540.6	2c	4352.8	5875.8
7c	4064.6	5540.6	2d	4352.8	5875.8
						7d	4064.6	5540.6	2′-1	4352.8	5875.8
4f	4065.6	5541.6	2′-2	4352.8	5875.8
						4e′	4064.6	5556.6	3a	4352.8	5875.8
4f	4065.6	5557.6	3b	4352.8	5875.8
						4g	4065.6	5557.6	3d	4352.8	5875.8
5a	4080.6	5525.6	3e	4352.8	5875.8
						3b	4080.6	5541.6	4c	4352.8	5875.8
4b	4080.6	5541.6	4d	4352.8	5875.8
						4c	4080.6	5541.6	4f	4352.8	5875.8
4d	4080.6	5541.6	4f	4352.8	5875.8
						4e	4080.6	5541.6	4g	4352.8	5875.8
4h	4080.6	5541.6	6a	4352.8	5875.8

4k	4080.6	5541.6	6b	4352.8	5875.8
						2d	4088.6	5515.6	7c	4353.8	5876.8
2b	4088.6	5530.6	1b	4368.8	5860.8
						3f	4096.6	5526.6	1f	4368.8	5860.8
2a	4104.6	5500.6	3c	4368.8	5860.8
						2c	4104.6	5500.6	3f	4368.8	5860.8
2e	4104.6	5500.6	4a	4368.8	5860.8
						2′	4104.6	5500.6	4e	4368.8	5860.8
2′	4104.6	5500.6	4h	4368.8	5860.8
						3a	4111.6	5510.6	4k	4368.8	5860.8
3c	4111.6	5510.6	5a	4368.8	5860.8
						3d	4111.6	5510.6	7a	4368.8	5860.8
3e	4111.6	5510.6	7b	4368.8	5860.8
									7d	4368.8	5860.8

TABLE 4

3d	2200.4	4044.6	7c	2144.4	4125.6	1b	4272.8	4368.8
									4g	2200.4	4044.6	7d	2144.4	4125.6	1c	4272.8	4368.8
4k	2200.4	4053.6	3a	2159.4	4078.6	1e	4272.8	4368.8
									3b	2200.4	4069.6	3c	2159.4	4078.6	4a	4272.8	4368.8
3c	2200.4	4069.6	3d	2159.4	4078.6	4e′	4272.8	4368.8
									3e	2200.4	4069.6	3e	2159.4	4078.6	7a	4272.8	4368.8
4e	2206.4	4037.6	3b	2159.4	4094.6	7b	4272.8	4368.8
									1b	2206.4	4062.6	3f	2159.4	4094.6	3d	4570.0	4744.0
1c	2206.4	4062.6	4c	2159.4	4094.6	3f	4546.0	4744.0
									2′-2	2206.4	4062.6	4d	2159.4	4094.6	3b	4272.8	4384.8
4f	2206.4	4062.6	4e	2159.4	4094.6	4b	4272.8	4384.8
									5a	2206.4	4062.6	4f	2159.4	4094.6	4c	4272.8	4384.8
1b	2215.4	4053.6	4h	2159.4	4094.6	4d	4272.8	4384.8
									2a	2215.4	4053.6	4k	2159.4	4094.6	4f	4272.8	4384.8
2b	2215.4	4053.6	4a	2159.4	4109.6	5a	4272.8	4384.8
									2c	2215.4	4053.6	4e′	2159.4	4109.6	4e	4296.8	4384.8
2d	2215.4	4053.6	4b	2184.4	4070.6	4g	4586.0	4714.0
									2e	2215.4	4053.6	4g	2184.4	4070.6	4k	4287.8	4384.8
2′-1	2215.4	4053.6	2a	2193.4	4061.6	4f	4562.0	4384.8
									3f	2215.4	4053.6	2b	2193.4	4061.6	4h	4562.0	4384.8
4c	2215.4	4053.6	2e	2193.4	4061.6	6a	4586.0	4698.0
									4d	2215.4	4053.6	2d	2193.4	4076.6	6b	4586.0	4698.0
4f	2215.4	4053.6	2c	2209.4	4046.6	7d	4586.0	4698.0
									4h	2215.4	4053.6	2′-1	2209.4	4046.6	1b	4288.8	4353.8
3a	2216.4	4054.6	2c	2209.4	4030.6	7c	4288.8	4353.8
									1d	2215.4	4078.6

Industrial applicability

In one embodiment of the present invention, misanalysis caused by errors in conventional methods for detecting mutations can be identified, and various mutations of 32 or more adjacent bases can be simultaneously detected. When individuals having mutations have various genotypes, it is possible to distinguish whether mutations at different sites are present in one genotype at the same time or present in a mixture of two or more genotypes. In addition, mutations resulting from gene deletions or insertions can be detected.

Thus, the present invention has been described in detail. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Claims (1)

1. A method of detecting a mutation, comprising:

a) amplifying a target polynucleotide using a primer pair to produce an amplified target polynucleotide, the primer pair selected from the group consisting of: SEQ ID NOS.2 and 3 for detecting a base mutation at position 2741 of the 4 th intron of the human maspin gene; SEQ ID NOS.7 and 8 for detecting a 3597 th base mutation of the 4 th intron of the human maspin gene; SEQ ID NOS.12 and 13 for detecting base mutations at the tyrosine-aspartic acid site of the hepatitis B virus DNA polymerase; SEQ ID NOS.20 and 21 for detecting base mutations in the 5' non-coding region of hepatitis C virus; SEQ ID NOS.25 and 26 for detecting base mutations in the 5' non-coding region of hepatitis C virus; and SEQ ID NOS.30 and 31 for detecting base mutations of the 5' non-coding region of hepatitis C virus;

b) contacting the amplified target polynucleotide with restriction endonucleases FokI and BstF5I, or restriction endonucleases MmeI and AvaII, to generate two or more restriction enzyme fragments each consisting of two single-stranded fragments of 2-32 nucleotides, wherein each of the single-stranded fragments comprises at least one mutated sequence which is a substitution, deletion or insertion of a base; and

c) detecting the molecular weight of the single-stranded fragment,

wherein the method is used for non-disease diagnostic purposes.