KR101025450B1 - Method for detecting multiple base mutations - Google Patents

Abstract

The present invention relates to a method for accurately and efficiently examining genetic nucleotide variations in living organisms. Specifically, a) amplifying a specific polynucleotide using multiple sequence primers (MSPs) and external primers (FPs); b) cutting the amplified specific polynucleotides with restriction enzymes to generate two or more single-stranded polynucleotide fragments containing a mutation sequence having a size of 2 to 32 bases; And c) measuring the molecular weight of the cleaved fragments.

Base Variation, Multiple Sequence Primer, External Primer, Annealing Temperature

Description

Method for detecting multiple base mutations

The present invention relates to a method for analyzing a gene of a living being. In particular, the present invention relates to a method for accurately and efficiently examining gene base mutations.

Genetic analysis of living organisms is widely used in relation to the risk of disease, diagnosis, prediction or treatment. For example, mutation analysis of specific genes of a specific person may lead to preventive efforts by predicting the degree of disease risk. In addition, genetic variation analysis of other organisms causing infections, such as viruses, can be used to induce effective treatment by examining whether the virus is resistant to therapeutic drugs.

Interest in genetic analysis has been heightened by the possibility that the human genome map can be completed to provide a broader estimate of disease risk, diagnose or predict disease, and predict drug response. However, in order to clarify the relationship between genetic variation of humans and other diseases and diseases, a large number of sequencing information should be analyzed as well as accurate and effective analysis of sequence variation.

DNA sequencing is a traditional method for analyzing nucleotide sequences or variations in bases. DNA sequencing includes the Maxam-Gilbert procedure and the Sanger procedure, but the latter method is mainly used. This method can analyze at least hundreds of nucleotide sequences in one reaction, which is effective for extensive sequencing or screening of mutations, but inefficient when examining only genetic mutations at specific positions. By providing the surrounding sequences that do not need to be examined at the same time, it is rather unnecessary to interpret only the mutations to be investigated, and in the case of some sequences, accurate sequence information can be obtained according to the physicochemical characteristics of the site. There may be cases where there are no similar sequences, or where similar sequences exist at multiple positions or when information is provided at a location other than the desired position. In addition, when there are several kinds of mutations in the same location, such as a virus infected with the human body, there is a problem that only provides the most information of the kind, and does not properly provide a small number of mutations.

Techniques developed to efficiently analyze base mutations at specific sites include PCR-SSCP (Polymerase chain reaction-Single stranded conformation polymorphism), allele specific hybridization, oligo-ligation, mini Mini-sequencing and RFMP. A method using a DNA chip has also been introduced, which is in principle no different from allel specific hybridization, except that it is differentiated from a stationary phase to which an oligonucleotide probe is attached.

PCR-SSCP (Orita, M. et.al, Genomics, 1989, 5: 8874-8879) amplifies the sequences containing the bases to be analyzed by PCR, separates them into their respective chains, and then polyacrylamide gels. Perform electrophoresis at. Since the secondary structure of the DNA chain is changed by one base difference, the base variation is examined according to the electrophoretic migration speed caused by this difference.

Allel specific hybridization is a method of hybridizing a sample DNA labeled with a radioisotope to a probe attached to a nylon filter or the like, and examining whether or not there is a base mutation by controlling conditions such as temperature and hybridization.

The oligol ligation method (Nucleic Acid Research 24, 3728, 1996) investigates base mutations by performing reactions under conditions where ligation does not occur in a condition that is not complementary to template DNA and checking whether the ligation proceeds. That's how.

Mini sequencing (Genome Research 7: 606, 1997) is designed to condition only one base at the position to be examined for polymerization and to detect different reactions depending on which one base is polymerized. This method was developed to analyze the base variation of.

PCR-SSCP, allel specific hybridization, oligo ligation methods are inconvenient to analyze large amounts of samples using polyacrylamide gels or fail to identify errors caused by the probes failing to bind to the desired location. There is this.

Minisequencing is convenient for analysis and advantageous for analyzing large amounts of samples, but still has the disadvantage of not identifying false results due to errors, and has limitations in that it cannot detect deletion and insertion of bases.

RFMP method (Korean Patent Nos. 642829 and 477766) is a technology developed to compensate for the above disadvantages, and generates two or more single-stranded polynucleotides containing a mutated sequence having a number of bases of 32 or less, and thus the molecular weight thereof. How to measure. The RFMP method allows easy and fast base mutation investigation while verifying the errors that can be generated by binding the primer to the wrong position, enabling accurate base mutation investigation and simultaneously shifting multiple adjacent bases within 32 base ranges. Although it is possible not only to investigate but also to detect genetic variation due to deletion or insertion, there is a disadvantage that the range of investigation in one reaction is limited to 32 bases. In other words, to examine all the different base variants over 32 bases apart, each base variant must be analyzed separately. That is, the restriction enzyme cleavage and the mass measurement process, including the PCR process, must all proceed as separate reactions.

It is a problem with most of the above-described base mutation analysis methods, except sequencing, to perform several reactions in order to analyze several base mutations that are separated from each other. For example, mutations due to drug resistance of the virus or mutation analysis for gene identification may not be determined by only one genetic mutation. In the case of adefovir prescribed for hepatitis B, drug resistance may occur simultaneously in genes that determine amino acids, such as the 181th and 236th, instead of a single mutation of the polymerase gene region of the virus. This should be judged accurately because it has a great influence on the future treatment direction of hepatitis patients. However, if each gene mutation is performed several times with each analysis, it is time-consuming and expensive, and a breakthrough technique is needed to solve this problem.

In order to solve the above problems, the primers can verify errors caused by binding to the wrong positions and detect genetic mutations due to deletion or insertion, thereby enabling accurate base mutation investigation and at the same time. An object of the present invention is to provide a method for analyzing multiple base mutations in which multiple base variants separated by one reaction can be examined.

The present invention to solve the above problems, a) amplifying a specific polynucleotide using a multi-sequence primer (MSP) and the outer primer (FP); b) cutting the amplified specific polynucleotides with restriction enzymes to generate two or more single-stranded polynucleotide fragments containing a mutation sequence having a size of 2 to 32 bases; And c) measuring the molecular weight of the cleaved fragments.

In the present invention, by using multiple sequence primers (MSP) and external primers (FP) in the amplification step of the polynucleotide, it is possible to analyze base mutations that are separated from each other at the same time, so that the base mutation investigation can be performed easily and quickly. Errors that can be generated by binding to the wrong position can be verified, and genetic variations due to deletions or insertions can be detected, enabling accurate investigation of base mutations. Therefore, the present invention can be used in various fields, such as the bio, medical and pharmaceutical fields that require analysis of base variations.

The above objects, features and advantages will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. Hereinafter, the present invention will be described in detail.

Justice

Definitions of terms used in the present invention are as follows.

In the present invention, "Multiple Sequence Primer (MSP)" means a primer including an external sequence, a restriction enzyme recognition sequence and a primer binding sequence.

In the present invention, "foreign sequence" means a sequence that does not exist in the template to be analyzed for the base mutation.

In the present invention, "foreign primer (FP)" means a primer including only an external sequence.

Detailed description of the invention

The present invention comprises the steps of a) amplifying a specific polynucleotide using multiple sequence primers (MSPs) and external primers (FPs); b) cutting the amplified specific polynucleotides with restriction enzymes to generate two or more single-stranded polynucleotide fragments containing a mutation sequence having a size of 2 to 32 bases; And c) measuring the molecular weight of the cleaved fragments.

In the present invention, by using multiple sequence primers (MSPs) and external primers (FPs) as primers, the genetic variations of living organisms can be easily and quickly investigated, while accurately and efficiently analyzing them, without separately analyzing multiple base mutations apart from each other. In addition to investigating at the same time, genetic variation due to deletion or insertion can be detected. In addition, the present invention can distinguish between the presence of multiple genotypes in one organism at the same time, whether the base mutations at different positions are present in one genotype or mixed in different genotypes. For example, humans have two (one pair) chromosomes that contain the same genetic information, so if a genetic mutation occurs, there may be mutations in both chromosomes (homo), but only one chromosome It may happen (hetero). When two or more adjacent base variants are all hetero, both base variants may be present on one chromosome at the same time, but may also be present on different chromosomes. The effects of two cases on life can be different and need to be distinguished. In the case of a virus that infects humans, the resistance of the prescribed drug can be tested to determine whether a mixture of species and wild type mutated in specific amino acids of HBV polymerase is present. In addition, when a series of drugs that may cause two or more resistances are sequentially administered, various base mutations may occur at different locations within one type of virus, and according to the present invention, this may be detected by one diagnostic analysis. Can be.

In addition, according to the present invention, it is possible to verify an error that the primer can generate by binding to the wrong position. If a primer binds to the wrong position, it is difficult to identify the error by analyzing only one single-stranded nucleotide, but the error can be identified by simultaneously analyzing other single nucleotides that are partially complementary. Thus, when analyzing two or more single-stranded nucleotides containing a mutated sequence together as in the present invention, it is possible to verify the errors that the primers can generate by binding to the wrong position.

In order to analyze the genetic variation, the present invention amplifies a desired nucleotide sequence so that the amplified product includes a site that can be cleaved with a restriction enzyme, so that the number of bases of the fragment after cleavage by the restriction enzyme is 32 or less. At least one of these bases is intended to be produced by replication of a template rather than a primer. In particular, the present invention, after digesting the amplified product with restriction enzymes, analyzes the genetic variation by measuring the molecular weight of each fragment, but simultaneously using two or more base mutations separated from each other by simultaneously using MSP and FP as primers. Allows simultaneous analysis. In a preferred embodiment of the present invention, by simultaneously using MSP and FP as primers in the analysis of genetic variation, two or more base mutations separated from each other by more than 32 bases can be analyzed simultaneously in one reaction.

In order to achieve the desired effect of the present invention, the external sequence used in the present invention is preferably made of 10 to 30 bases, most preferably made of 15 to 25 bases, but is not limited thereto. Calculating from the total amount of human genome gene, there is a possibility that somewhere in the human gene up to 17mer (the 17th power combination of 4) may have the same sequence. In the case of PCR, 17mer is sufficient in theory because it has to react with two kinds of primers, but primers before and after 20mer are generally used.

In a preferred embodiment of the present invention, 20mer which does not bind to any of human or virus genome can be determined as an external sequence and inserted into MSP and FP. The possibility of binding external sequences to human or viral genomes is determined by predicting mismatching using Needleman and Wunsch's sequencing techniques. Examples of external sequences that can be identified using Needleman and Wunsch's sequencing techniques are shown in Table 1 below, but the external sequences are not limited thereto, and any sequences not present in the template to be amplified are all external sequences according to the present invention. It can be used as. As long as it is not an analysis object, the sequence from plants, bacteria, etc. may be sufficient.

In one embodiment of the present invention, it is possible to perform a multi-base mutation analysis using the external sequence shown in Table 1 below, the results of the multi-base mutation analysis is the same using any external sequence shown in Table 1.

Example of external sequence External sequence Sequences (5'-3 ') One TTAAAGAGCGCTCCAAAGCC 2 GACGACGCCTCTCCTTTCCT 3 GTCCGTCCCTTAAGGTGGTT 4 GAGGTTCTGGGCGCGACTTT 5 CTACCAAGTCGCCGAACACA 6 AACATCCCGGCCCAGCAGGT 7 CAATGCCGTGGAACCTGAAA 8 TTAGAACGCCTTTAGCAGCC

In order to analyze multiple nucleotide variations, MSP and FP used in the present invention amplify a desired base sequence to include a site where a PCR amplification product can be cleaved with a restriction enzyme, but generate a fragment having a structure as shown in the following figure. . MSP consists of an external sequence, a restriction enzyme recognition sequence and a primer binding sequence, in the present invention, the MSP is used as two types of pairs of a forward primer and a reverse primer, the forward primer is an external sequence 1, restriction enzyme recognition sequence 1 and a primer binding sequence 1, wherein the reverse primer comprises an external sequence 2, restriction enzyme recognition sequence 2 and primer binding sequence 2. In the present invention, the FP is also used as two types of pairs of a forward primer and a reverse primer, the forward primer includes an external sequence 1, and the reverse primer includes an external sequence 2.

<PCR amplification product before restriction enzyme treatment>

5'- External sequence 1 Restriction enzyme
Cognitive Sequence 1 primer
Combined Sequence 1 Before mutation
order Variant sequence After variation
order primer
Combined Sequence 2 Restriction enzyme
Cognitive Sequence 2 External sequence 2
-3 '

The "restriction enzyme recognition sequence" is a sequence recognized by restriction enzymes, and may not match the sequence to be cleaved. For example, Fok1 recognizes the GGATG sequence but the cleaved position is the base after the ninth base from the 3 'end of the recognition sequence.

According to the present invention, the PCR amplification products before the restriction enzyme treatment are generated in pairs as many as the number of pairs of MSPs used, which are each assay site in which there are base mutations separated by at least 32mer from the entire base sequence of analysis. This is because an appropriate MSP primer pair is prepared and used separately. In addition, since the restriction enzyme recognition sequence is introduced and used for each MSP primer, the pair of PCR amplification products is also made of a sequence including the restriction enzyme recognition sequence. Thereby, according to the present invention, several base mutations separated from each other can be irradiated by one PCR amplification.

A fragment generated after cleavage by the restriction enzyme of the PCR amplification product includes a mutant sequence, and its structure is shown in the following figure.

<Section after restriction enzyme treatment of PCR amplification product>

5'- primer
Combined Sequence 1 Before mutation
order Variant sequence After variation
order primer
Combined Sequence 2 -3 '

The number of bases of the fragments can be determined according to the purpose of multiplexing, using the principle that the products to be cut vary according to the insertion position of the restriction enzyme recognition sequence. Restriction enzyme recognition sequence 1 is a sequence inserted into the forward primer, restriction enzyme recognition sequence 2 is a sequence inserted into the reverse primer. The primer binding sequence is a sequence capable of complementarily binding to a nucleic acid as a template, but the restriction enzyme recognition sequence may not be complementary to the nucleic acid.

In order for the primer to bind to template DNA, the number of bases of the primer binding sequence should be at least 4 or more, preferably 8 or more and 30 or less, but is not limited thereto. "Previous sequence" is the 5 'side sequence of the variant to be investigated, the number of bases should be 0 to 31. A "mutation sequence" is a sequence containing a mutated base to be investigated, derived from a template, and having a base substitution, insertion, deletion, etc., and the number of mutant bases is usually one or more than two. . "Post-mutation sequence" is a sequence on the 3 'side of the mutation sequence to be investigated, the number of bases should be 0 to 31.

The fragments generated after restriction enzyme cleavage should include a mutant sequence, and the number of bases of the fragments is preferably 2 to 32, more preferably 6 to 16, but is not limited thereto. This is when the analysis by mass spectrometry, the analysis results reflect the size of the good section. If the fragment consists of only one base, it contains only the mutant sequence, so it is difficult to confirm whether the primer binds to the wrong position, which is not preferable.

In the present invention, MSP and FP are put together in the reactant, but MSP is added in a small amount. As shown in Figure 1 in the initial PCR reaction (2 ~ 15Cycle) by setting the annealing (annealing) temperature suitable for the primer binding sequence of the MSP, after a small amount of MSP injected to generate a small amount of PCR amplification target for the template Most of it is exhausted (see Figure 1.A.). After the initial reaction, when the temperature is set to an annealing temperature suitable for the FP, several different mutations are separated from each other using the PCR amplification product produced by the initial reaction as a primer as a new template (see Figure 1.B.). PCR amplifications containing each base are evenly generated (see Figure 1.C.). In order to carry out the present invention, the annealing temperature of the primer binding sequence of the MSP and the annealing temperature of the FP should be different. Therefore, it should be noted that the annealing temperature of the primer binding sequence of the MSP and the annealing temperature of the FP are not the same in the preparation of the MSP and FP. Annealing temperature suitable for the primer binding sequence of the MSP is preferably 40 ℃ to 55 ℃, but is not limited to this may vary depending on the primer binding sequence used. In addition, the annealing temperature suitable for FP is preferably from 60 ℃ to 70 ℃, but is not limited to this may vary depending on the FP used. The number of PCR cycles for MSP exhaustion at the beginning of the PCR reaction is preferably 2 to 15 times, more preferably 5 to 12 times, but is not limited thereto and may vary depending on reaction conditions. The amount of MSP added to the reactant in order to exhaust the MSP at the beginning of the PCR reaction is preferably 0.03 μM to 0.15 μM, more preferably 0.05 μM to 0.1 μM, but is not limited thereto and may also vary depending on the reaction conditions.

Example

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, but it should be noted that the contents of the present invention are not limited thereto.

Example 1 Base Variation Analysis of Different Sites of GJB2 (gap junction protein beta 2) Gene

Base mutations in several different regions of GJB2, known to determine human innate deafness, were investigated (NM_004004; chromosome 13). Example 1 is performed on human genome DNA.

One. PCR  Amplification

The sequence (5 '→ 3') of the template DNA of GJB2 to be analyzed is as follows.

GATTGGGGCACGCT GCAGACGATCCTGGGG GgT GTGAACAAACACTCCAC CAGCATTGGAAAGATCTGGCTCACCGTCCTCTTCATTTTTCGCATTATGATCCTCGTTGTGGCTGCAAAGGAGGTGTGGGGAGATGAGCAGGCCGACT TTGTCTGCAACACC CtG CAGCCAGGCTGCAAGAA CGTGTGCTACGATCACTACTTCCCCATCTCCCACA TCCGGCTATGGGC CcT GCAGCTGATCTTCGT GTCCACGCCAGCGCTCCTAGTG (Sequence Information 1)

The underlined sequences are the sites where primers 1 and 2, primers 3 and 4, and primers 5 and 6 bind to each other by primer pairing. Bases written in lowercase are 'sequence sequences'.

Primer 1. 5'- TTAAAGAGCGCTCCAAAGCCggatgGCAGACGATCCTGGGG-3 '(41mer) (SEQ ID NO: 2)

Primer 2. 5'- GACGACGCCTCTCCTTTCCTggatgTGGAGTGTTTGTTCAC-3 '(41mer) (SEQ ID NO: 3)

Primer 3. 5'- TTAAAGAGCGCTCCAAAGCCggatgTTGTCTGCAACACC-3 '(39mer) (SEQ ID NO: 4)

Primer 4. 5'- GACGACGCCTCTCCTTTCCTggatgTTCTTGCAGCCTGGCTG-3 '(42mer) (SEQ ID NO: 5)

Primer 5. 5'- TTAAAGAGCGCTCCAAAGCCggatgATCCGGCTATGGGC-3 '(39mer) (SEQ ID NO: 6)

Primer 6. 5'- GACGACGCCTCTCCTTTCCTggatgACGAAGATCAGCTGC-3 '(40mer) (SEQ ID NO: 7)

The primers 1 to 6 are MSPs, primers 1, 3 and 5 are forward primers, and primers 2, 4 and 6 are reverse primers. As described above, MSP primers 1 to 6 each include an external sequence, a restriction enzyme recognition sequence, and a primer binding sequence. Lowercase sequences are the recognition sequences of Fok1.

In addition to the primers 1 to 6, the following primers 7 and 8 were added as FP. Primer 7 is a forward primer and Primer 8 is a reverse primer. As described above, primers 7 and 8, which are FP, contain only external sequences.

Primer 7. 5'- TTAAAGAGCGCTCCAAAGCC-3 '(20mer) (SEQ ID NO: 8)

Primer 8. 5'- GACGACGCCTCTCCTTTCCT-3 '(20mer) (SEQ ID NO: 9)

PCR buffer (1x), MgSO ₄ 2 mM, dNTP 200 mM, Platinum Taq Polymerase (Invitrogen, 10966-026) 0.5 U, primers 1 and 2, 3 and 4, and 5 0.05 μM and 6, 0.4 μM of primers 7 and 8, 100 ng of genome DNA, respectively, and adjust the total reaction volume to 25 μl. And PCR reaction is performed under the following conditions.

5 minutes at 94 ° C,

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

94 ℃ 15 seconds 62 ℃ 15 seconds 72 ℃ 15 seconds (40 cycles),

72 ℃ 5 minutes

DNA is extracted from blood and purely isolated according to conventional methods. For example, from the blood using the 'SDS / Protease K' method (Maniatis, Molecular Cloning, A laboratory Manual, Cold Spring Harber Laboratory Press, Cold Spring Harbor, 1989) or QIAamp DNA Mini Kit 250 (Qiagen 51106). DNA can be isolated. If blood is tested on paper, follow the Dried Blood Spot protocol of the QIAamp DNA Mini Kit. If the DNA concentration is low, the DNA can be concentrated and used in the following manner. Add 1/10 volume of 3M sodium acetate (pH 5.3) and 2.5 volumes of ethanol to the DNA solution, gently mix, and leave at -20 ℃ for 1 hour or more. After centrifugation at 4 ℃, 13000 rpm for 15 minutes. Carefully remove the supernatant, add 70% ethanol, and centrifuge at 13000 rpm for 10 minutes at 4 ° C. After drying the ethanol, the desired volume of distilled water is added to dissolve.

The sequence of fragments generated by PCR is as follows. (5 '-> 3').

The product amplified by primers 1 and 2 to the template

TTAAAGAGCGCTCCAAAGCCggatgGCAGACGAT CCTGGGGG [G /-] TGTG AACAAACACTCCAcatccAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 10) and

AATTTCTCGCGAGGTTTCGGcctacCGTCTGCTAGGAC CCCC [C /-] ACACTTGT TTGTGAGGTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 11),

The product amplified by the primers 3 and 4 to the template

TTAAAGAGCGCTCCAAAGCCggatgTTGTCTGCA ACACCC [T /-] GCAG CCAGGCTGCAAGAA catccAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 12) and

AATTTCTCGCGAGGTTTCGGcctacAACAGACGTTGTG GG [A /-] CGTCGGTC CGACGTTCTTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 13),

The product amplified by the primers 5 and 6 to the template

TTAAAGAGCGCTCCAAAGCCggatgTCCGGCTAT GGGCC [C /-] TGC AGCTGATCTTCGTcatccAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 14) and

AATTTCTCGCGAGGTTTCGGcctacAGGCCGATACCCG G [G /-] ACGTCGA CTAGAAGCAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 15).

The products amplified by primers 7 and 8 to the template are the same as the products amplified by primers 1 and 2, 3 and 4, and 5 and 6 into the template. In this case, the appropriate MSP primer pair was separately prepared and used for each analysis site in which there are base mutations separated by at least 32 mer from the base sequences. As a result, the PCR amplification product was not composed of one amplification product for the entire template sequence of SEQ ID NO. 1, but was composed of three pairs of amplification products each containing three kinds of base variants separated from each other. Further, by introducing and using a restriction enzyme recognition sequence for each MSP primer, the three pairs of amplification products thus obtained were also made into sequences each containing a restriction enzyme recognition sequence. As a result, according to the present invention, multiple base mutations separated from each other can be examined by one PCR amplification. The same applies to the following examples.

The region indicated by lowercase letters in the sequence generated by the PCR is the restriction enzyme recognition sequence, the underlined region is the sequence of the fragment generated by cleavage by the restriction enzyme, and the region indicated by the square brackets ([]) is "variable sequence" to be.

2. Cleavage, Purification by Restriction Enzymes and Desalination

To the product amplified by PCR, FokI (NEB R109L) 1 U, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 1 mM DTT (PH 7.9 at 25 ° C) The reaction is carried out at 37 ° C. for 1 hour.

After purely separating the DNA fragments cleaved from the solution treated with the restriction enzyme, it is preferable to measure the molecular weight of the separated DNA fragments. For example, Oasis (Waters) C18 ion resin exchange column (C18 reverse phase column chromatography) can be used for pure separation of DNA fragments cut by restriction enzymes. First, 70 μl of 0.15M triethylammonium acetate (TEAA, pH 7.6) is added to the solution treated with the restriction enzyme and placed for 1 minute. Resin was activated by passing 1 ml of 100% acetonitrile (ACN; Sigma, USA) and 1 ml of 0.1 M TEAA, and then 100 µl of a mixed solution of the restriction enzyme-treated solution and 0.15 M TEAA, 2 ml of 0.1 M TEAA and 1 ml of tertiary distilled water are passed through in turn. Place the column on a Collection Plate and pass 100 μl of 70% ACN. Once the eluate is collected in the collection plate, the collection plate is dried at 120 ° C. for 60 minutes.

3. Maldi- Toffee mass  Spectrometry ( MALDI - TOF Mass Spectrometry )

4 μl of the Maldi matrix (22.8 mg ammonium citrate, 148.5 mg hydroxypicolinic acid, 1.12 mL acetonitrile, 7.8 mL H ₂ O) was added to the Maldi-tope mass spectrometry (Biflex IV, After dotting in Bruker's anchor chip plate in advance, it is dried for 30 minutes at 37 ℃. 10 μl of tertiary distilled water was dissolved in the sample of the collection plate which had been purified and desalted, and then 2 μl of the dried maldi matrix was dried, and the maldi matrix was dried again at 37 ° C. for 30 minutes, and then the maldi-tope mass was removed. Analyze with spectrometry. The analytical method follows the manual of Maldi-Top Mass Spectrometry.

When the 35th base of the GJB2 gene is normal (G), the molecular weights of the fragments generated after cleavage by the restriction enzyme are 4141.6 D and 3910.6 D (14mer), and when the 167th base of the GJB2 gene is normal (T), the restriction enzyme The molecular weights of the fragments generated after cleavage were 3655.4 D and 3742.4 D (12mer), and when the 235th base of the GJB2 gene was normal (C), the molecular weights of the fragments generated after cleavage by the restriction enzyme were 3100 D and 3173 D. (10 mer). When the 35th base of the GJB2 gene is deleted (-T), the molecular weights of the fragments generated after cleavage by restriction enzymes are 3813.4 D and 3621.4 D (13mer), and when the 167th base of the GJB2 gene is deleted (-T). ), The molecular weights of the fragments generated after cleavage by restriction enzymes are 3351.2 D and 3429.2 D (11mer), and when the 235th base of the GJB2 gene is deleted (-G), the molecular weight of the fragment generated after cleavage by the restriction enzyme Are 2810.8 D and 2843.8 D (9mer). Therefore, in the case of heterotypes, all the molecular weights of the top and the deletion of the base are shown.

As shown in Figure 2, using the analysis method according to the present invention can determine whether the base mutation of three different regions on the GJB2 gene as a single diagnostic analysis results. 3 shows the case where the 235th base of the GJB2 gene is hetero.

Example 2. PDS2 (Pendred syndrome 2) another base mutation analysis of other regions of the gene.

Nucleotide mutations in several different regions of PDS2, another gene known to determine human innate deafness, were investigated (MIM605646; 7th chromosome). Example 2 was carried out by a similar method as in Example 1.

One. PCR  Amplification

The sequence (5 '→ 3') of the template DNA of PDS2 to be analyzed is as follows. This Example 2 aims to detect seven base variants at the same time. Since the seven base variants are separated from each other on the chromosome, sequence information is described as follows.

SNP: 2162C> T & 2168A> G

AGGACA CATTCTTTTTGA cGGTCCa TGATGCTATACT CTATCTACAGAAC (SEQ ID NO: 16)

SNP: IVS7-2A> G

ACATC TTTTGTTTTATTTC aGACG ATAATTGCTACTGC CATTTCA (SEQ ID NO: 17)

SNP: IVS9 + 3A> G

CCAT CGATGGGAACCAGGT aTGG GTGCCCTTTTGCTGA ACTGGTT (SEQ ID NO: 18)

SNP: 439A> G

TTTT CCAGTGGTGAGTTTA aT GGTGGGATCTGTTGT TCTGAGCAT (SEQ ID NO: 19)

SNP: 365insT

GTC TCTACTCTGCTTTTT tCcCT ATCCTGACATACTTT ATCTTT (SEQ ID NO: 20)

The underlined sequences are the sites where 9 and 10, 11 and 12, 13 and 14, 15 and 16, and 17 and 18 bind in primer pairs. Bases written in lowercase are 'sequence sequences'.

Primer 9. 5 '-TTAAAGAGCGCTCCAAAGCCAGGACggatgCATTCTTTTTGA -3' (42mer) (SEQ ID NO: 21)

Primer 10. 5 '-GACGACGCCTCTCCTTTCCTAGATAggatgAGTATAGCATCA -3' (42mer) (SEQ ID NO: 22)

Primer 11. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTTTTGTTTTATTTC-3' (39mer) (SEQ ID NO: 23)

Primer 12. 5 '-GACGACGCCTCTCCTTTCCTggatgGCAGTAGCAATTAT -3' (39mer) (SEQ ID NO: 24)

Primer 13. 5 '-TTAAAGAGCGCTCCAAAGCCggatgCGATGGGAACCAGGT -3' (40mer) (SEQ ID NO: 25)

Primer 14. 5 '-GACGACGCCTCTCCTTTCCTggatgTCAGCAAAAGGGCAC -3' (40mer) (SEQ ID NO 26)

Primer 15. 5 '-TTAAAGAGCGCTCCAAAGCCggatgCCAGTGGTGAGTTTA -3' (40mer) (SEQ ID NO: 27)

Primer 16.5 '-GACGACGCCTCTCCTTTCCTggatgACAACAGATCCCACC -3' (40mer) (SEQ ID NO: 28)

Primer 17. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTCTACTCTGCTTTTT -3' (40mer) (SEQ ID NO: 29)

Primer 18. 5 '-GACGACGCCTCTCCTTTCCTggatgAAAGTATGTCAGGAT -3' (40mer) (SEQ ID NO: 30)

The primers 9 to 18 are MSPs, primers 9, 11, 13, 15 and 17 are forward primers, and primers 10, 12, 14, 16 and 18 are reverse primers. As described above, MSP primers 9 to 18 each include an external sequence, a restriction enzyme recognition sequence, and a primer binding sequence. Lowercase sequences are the recognition sequences of Fok1.

Experimental methods including PCR reactions put primers 9 and 10, 11 and 12, 13 and 14, 15 and 16, and 17 and 18 in 0.05 μM and primers 7 and 8 in 0.4 μM, respectively. Same as Example 1. However, PCR conditions are performed as follows.

5 minutes at 94 ° C,

94 ℃ 15 seconds 43 ℃ 15 seconds 72 ℃ 15 seconds (10 cycles),

94 ℃ 15 seconds 62 ℃ 15 seconds 72 ℃ 15 seconds (40 cycles),

72 ℃ 5 minutes

The sequence of fragments generated by PCR is as follows (5 '-> 3').

The products amplified by primers 9 and 10 to the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCATTCTTTT TGA [C / T] GGTCC [A / G] TGATGCTATACTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 31) and

AATTTCTCGCGAGGTTTCGGcctacGTAAGAAAAACT [G / A] CCAGG [T / C] ACT ACGATATGAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 32),

The products amplified by primers 11 and 12 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTTTTGTTTT ATTTC [A / G] GACGA TAATTGCTACTGCcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 33) and

AATTTCTCGCGAGGTTTCGGcctacAAAACAAAATAAA G [T / C] CTGCTATTA ACGATGACG

gtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 34),

The products amplified by primers 13 and 14 to the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCGATGGGAA CCAGGT [A / G] TGGGT GCCCTTTTGCTGAcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 35) and

AATTTCTCGCGAGGTTTCGGcctacGCTACCCTTGGTC CA [T / C] ACCCACGGG AAAACGACTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 36),

The products amplified by primers 15 and 16 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCCAGTGGTG AGTTTA [A / G] TGG TGGGATCTGTTGTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 37) and

AATTTCTCGCGAGGTTTCGGcctacGGTCACCACTCAA AT [T / C] ACCACCC TAGACAACA

gtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 38),

The products amplified by primers 17 and 18 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTCTACTCTG CTTTTTT [-/ T] CC [C / T] TAT CCTGACATACTTTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 39) and

AATTTCTCGCGAGGTTTCGGcctacAGATGAGACGAAA AAA [-/ A] GGGAATAGGA CTGTATGAAAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 40),

The products amplified by primers 7 and 8 to the template are identical to the products amplified by primers 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18 into the template.

The region indicated by the lowercase letter in the sequence generated by the PCR is a restriction enzyme recognition sequence, the underlined portion is the sequence of the fragment generated by cleavage by restriction enzyme, the site indicated by brackets ([]) is the 'mutation sequence' to be.

The production principle of the amplification product is the same as in Example 1.

2. Cleavage, Purification by Restriction Enzymes and Desalination

It is carried out in the same manner as in Example 1.

3. Maldi- Sat F Mass Spectrometry

It is carried out in the same manner as in Example 1.

Of PDS2
Target SNP Analysis target
Zino Type In 5'-3 'mould
Amplified
Mass of the intercept (Da) In 3'-5 'mould
Amplified
Mass of the intercept (Da) 2162C> T &2168A> G
(9mer)

C & A 2794.8 2778.8 C & G 2794.8 2763.8 T & A 2809.8 2778.8 T & G 2809.8 2763.8 IVS7-2A> G
(11mer) A 3420.2 3402.2 G 3436.2 3387.2 IVS9 + 3A> G
(12mer) A 3781.4 3695.4 G 3797.4 3680.4 439A> G
(10mer) A 3162 3012 G 3178 2997 365insT /
(13 / 14mer)

No change & C 3921.6 4136.6 Insert T & C 4225.8 4449.8 No change & T 3936.6 4120.6 Insert T & T 4240.8 4433.8

As a result of analyzing the size of the fragments generated by the reaction by Maldi-Top mass spectrometry, the gene type of the PDS2 may be determined based on Table 2 above. For the hetero type all molecular weights of the base are shown.

As shown in Figures 4 and 5, using the analysis method according to the invention it can be determined whether the base mutation of the seven different sites on the PDS2 gene as a single diagnostic analysis results.

Example 3 Base Variation Analysis of Various Genetic Markers for Application to Forensic Science.

Genetic markers (SNP markers) capable of identifying specific genetic distributions of specific populations and using them to assess race (group, ethnicity) are applied to this analysis. This application to forensic science requires a wide range of genes known to be racially different, so RFMP multiplexing is a necessity. Example 3 uses, as a template, FUT2 (fucosyltransferase 2) and three ancestry-informative markers (AIMs), genes that characterize blood, having different allele frequencies by race. The FUT2 test genes located on chromosome 19 are two, 171th and 960th bases, three AIMs marker names are MID575, FY-NULL, and TYP-191, and NCBI database SNP numbers are rs140864, rs2814778 and rs1042602, respectively.

One. PCR  Amplification

The sequence (5 '→ 3') of the template DNA to be analyzed is as follows. Example 3 aims to detect five base mutations simultaneously. Since the five base mutation sites are separated from each other on the chromosome, sequence information is described as follows.

SNP: FUT2 A171G

TGGACGATCAATGC a ATAGGCCGCCTGGGGA (SEQ ID NO: 41)

SNP: FUT2 A960G

CCTGCCGGAGTGGAC a GGGATTGCCGCAGACC (SEQ ID NO: 42)

SNP: MID575 (rs140864)

ATGTCCAATCTGCTC ttc AAGTTCAACCAACCT (SEQ ID NO: 43)

SNP: FY-NULL (rs2814778)

CGCCTGTGCTTCCAAG a TAAGAGCCAAGGACTAA (SEQ ID NO: 44)

SNP: TYP-191 (rs1042602)

TGCACTGCTTGGGGGAT c TGAAATCTGGAGAGACA (SEQ ID NO: 45)

The underlined sequences are the sites where primers 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28 bind in primer pairs. Bases written in lowercase are 'sequence sequences'.

Primer 19. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTGGACGATCAATGC -3' (39mer) (SEQ ID NO: 46)

Primer 20. 5 '-GACGACGCCTCTCCTTTCCTggatgTCCCCAGGCGGCCTAT -3' (41mer) (SEQ ID NO: 47)

Primer 21 .5 '-TTAAAGAGCGCTCCAAAGCCggatgCCTGCCGGAGTGGAC -3' (40mer) (SEQ ID NO: 48)

Primer 22. 5 '-GACGACGCCTCTCCTTTCCTggatgGGTCTGCGGCAATCCC -3' (41mer) (SEQ ID NO: 49)

Primer 23. 5 '-TTAAAGAGCGCTCCAAAGCCggatgATGTCCAATCTGCTC -3' (40mer) (SEQ ID NO: 50)

Primer 24. 5 '-GACGACGCCTCTCCTTTCCTggatgAGGTTGGTTGAACTT -3' (40mer) (SEQ ID NO: 51)

Primer 25. 5 '-TTAAAGAGCGCTCCAAAGCCggatgCGCCTGTGCTTCCAAG -3' (41mer) (SEQ ID NO: 52)

Primer 26. 5 '-GACGACGCCTCTCCTTTCCTggatgTTAGTCCTTGGCTCTTA -3' (42mer) (SEQ ID NO: 53)

Primer 27. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTGCACTGCTTGGGGGAT -3' (42mer) (SEQ ID NO: 54)

Primer 28. 5 '-GACGACGCCTCTCCTTTCCTggatgTGTCTCTCCAGATTTCA -3' (42mer) (SEQ ID NO: 55)

The primers 19 to 28 are MSPs, primers 19, 21, 23, 25 and 27 are forward primers, and primers 20, 22, 24, 26 and 28 are reverse primers. As described above, primers 19 to 28, which are MSPs, include an external sequence, a restriction enzyme recognition sequence, and a primer binding sequence, respectively. Lowercase sequences are the recognition sequences of Fok1.

Experimental methods including PCR reactions put 0.05 μM for primers 19 and 20, 21 and 22, 23 and 24, 25 and 26, and 27 and 28, and 0.4 μM for primers 7 and 8, respectively. Same as Example 1.

The sequence of fragments generated by PCR is as follows (5 '-> 3').

The product amplified by the primers 19 and 20 to the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTGGACGATC AATGC [A / G] ATA GGCCGCCTGGGGAcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 56) and

AATTTCTCGCGAGGTTTCGGcctacACCTGCTAGTTAC G [T / C] TATCCGG CGGACCCCTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 57),

The products amplified by primers 21 and 22 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCCTGCCGGA GTGGAC [A / G] GGG ATTGCCGCAGACCcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 58) and

AATTTCTCGCGAGGTTTCGGcctacGGACGGCCTCACC TG [T / G] CCCTAAC GGCGTCTGGgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 59),

The products amplified by primers 23 and 24 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgATGTCCAAT CTGCTC [ TTC /-] AA GTTCAACCAACCTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 60) and

AATTTCTCGCGAGGTTTCGGcctacGACAGGTTAGACG AG [ AAG /-] TTCAAG TTGGTTGGAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 61),

The products amplified by primers 25 and 26 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCGCCTGTGC TTCCAAG [A / G] TAAG AGCCAAGGACTAAcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 62) and

AATTTCTCGCGAGGTTTCGGcctacGCGGACACGAAGG TTC [T / C] ATTCTCGG TTCCTGATTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 63),

The products amplified by primers 27 and 28 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTGCACTGCT TGGGGGAT [C / A] TGAA ATCTGGAGAGACAcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 64) and

AATTTCTCGCGAGGTTTCGGcctacACGTGACGAACCC CCTA [G / T] ACTTTAGA CCTCTCTGTgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 65).

The products amplified by primers 7 and 8 into the template are identical to the products amplified by primers 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28 into the template.

The region indicated by the lowercase letter in the sequence generated by the PCR is a restriction enzyme recognition sequence, the underlined portion is the sequence of the fragment generated by cleavage by restriction enzyme, the site indicated by brackets ([]) is the 'mutation sequence' to be.

The production principle of the amplification product is the same as in Example 1.

2. Cleavage, Purification by Restriction Enzymes and Desalination

It is carried out in the same manner as in Example 1.

3. Maldi- Sat F Mass Spectrometry

It is carried out in the same manner as in Example 1.

Target SNP Analysis Gino Type In 5'-3 'mold
Amplified
Mass of the intercept (Da) In 3'-5 'mold
Amplified
Mass of the intercept (Da) FUT2A171G
(9mer) A 2810.8 2809.8 G 2826.8 2794.8 FUT2A960G
(10mer) A 3213.0 3043.0 G 3229.0 3028.0 MID575 (rs140864)
(11 / 8mer) TTC + 3347.2 3469.2 TTC Deletion 2449.6 2513.6 FY-NULL (rs2814778)
(12mer) A 3733.4 3682.4 G 3749.4 3667.4 TYP-191 (rs1042602)
(13mer) C 4134.6 4013.6 A 4158.6 3988.6

As a result of analyzing the size of the fragments generated by the reaction by Maldi-Top mass spectrometry, the gene type of the SNP marker may be determined based on Table 3 above. For the hetero type all molecular weights of the base are shown.

As shown in Figure 5, using the analysis method according to the invention it can be determined whether the base mutation of the five different sites on the SNP marker gene as a single diagnostic analysis results.

Example 4 Analysis of base mutations in the site of resistance to entacarvir of hepatitis B virus DNA polymerase.

Nucleotide mutations appear in the DNA polymerase gene of hepatitis B virus that causes hepatitis B in humans. Base mutations in some of these sites develop resistance to entacavir, a therapeutic agent for hepatitis B, including amino acids I169T, S202G / I / C, M250V / I / L, and T184L / F / A / M / S / I / C / G and others have been reported as causative mutations.

One. PCR  Amplification

The sequence (5 '→ 3') of the template DNA to be analyzed is as follows. Example 4 aims to detect four base variants at the same time. Since the four base variants are separated from each other on the HBV genome, sequence information is described as follows. Each of the four base variants refers to a 169 th, 184 th, 202 th and 250 th codons, which in turn encode HBV polymerase amino acids.

ACTGCACTTGTATTCCCATCCCATCAT CCTGGGCTTTCGCAAG att CCTATGGGAGTG GGCCTCAGTCCGT TTCTCATGGCTCAGTTT act AGTGCCATTTGT TCAGTGGTTCGCAGGGCTTTCCCCCACT GTTTGGCTTTC agt atg TATATGGATGATGTGGTATTG GGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTCTGTCTTTGGGTATACATTTAAACCCTAATAAAACCAAGCGTTGGGGCTAC TCCCTTAACTTC GGATATGT TTGACCCCTAATAAAACCAAA (sequence information 66)

Underlined sequences are the sites where primers 29 and 30, 31 and 32, 33 and 34, and 35 and 36 bind in primer pairs. Bases written in lowercase are 'sequence sequences'.

Primer 29. 5 '-TTAAAGAGCGCTCCAAAGCCggatgCCTGGGCTTTCGCAAG -3' (41mer) (SEQ ID NO: 67)

Primer 30. 5 '-GACGACGCCTCTCCTTTCCTggatgCACTCCCATAGG -3' (37mer) (SEQ ID NO: 68)

Primer 31. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTTCTCCTGGCTCAGTTT -3' (42mer) (SEQ ID NO: 69)

Primer 32. 5 '-GACGACGCCTCTCCTTTCCTggatgACAAATGGCACT -3' (37mer) (SEQ ID NO: 70)

Primer 33. 5 '-TTAAAGAGCGCTCCAAAGCCggatgGTTTGGCTTTC -3' (36mer) (SEQ ID NO: 71)

Primer 34. 5 '-GACGACGCCTCTCCTTTCCTggatgCAATACCACATGATCCATATA -3' (46mer) (SEQ ID NO: 72)

Primer 35. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTCCCTTAACTTC -3' (37mer) (SEQ ID NO: 73)

Primer 36. 5 '-GACGACGCCTCTCCTTTCCTggatgAACTTCCAATTACATATCC -3' (44mer) (SEQ ID NO: 74)

The primers 29 to 36 are MSPs, primers 29, 31, 33 and 35 are forward primers, and primers 30, 32, 34 and 36 are reverse primers. As described above, primers 29 to 36, which are MSPs, include an external sequence, a restriction enzyme recognition sequence, and a primer binding sequence, respectively. Lowercase sequences are the recognition sequences of Fok1.

In the experimental method including the PCR reaction, primers 29 and 30, 31 and 32, 33 and 34, and 35 and 36 were put in 0.05 μM and primers 7 and 8, respectively, 0.4 μM. same. However, only PCR reaction is performed under the following conditions.

5 minutes at 94 ° C,

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

94 ℃ 15 seconds 62 ℃ 15 seconds 72 ℃ 15 seconds (35 cycles),

72 ℃ 5 minutes

DNA is extracted from the serum and purified purely according to conventional methods, and DNA can be separated from the serum using the QIAamp DNA Mini Kit 250 (Qiagen 51106). However, the amount of elution is determined separately from 40 to 100 μl, and the obtained DNA is not concentrated.

The sequence of fragments generated by PCR is as follows (5 '-> 3').

The products amplified by primers 29 and 30 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgCCTGGGCTT TCGCAAG [ AT T] CCTATGGGAGTGcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 75) and

AATTTCTCGCGAGGTTTCGGcctacGGACCCGAAAGCG TTC [ TAA ] GGA TACCCTCAC

gtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 76),

The products amplified by primers 31 and 32 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTTCTCCTGG CTCAGTTT [ AC T] AGTGCCATTTGTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 77) and

AATTTCTCGCGAGGTTTCGGcctacAGAGGACCGAGTC AAA [ TGA ] TCAC GGTAAACAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 78),

The product amplified by the primers 33 and 34 to the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgGTTTGGCTT TC [ GGA ] TATATGGA TCATGTGGTATTGcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 79) and

AATTTCTCGCGAGGTTTCGGcctacCAAACCGAAAG [C CT ] ATATACCTAGT ACACCATAACgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 80),

The product of primers 35 and 36 amplified against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTCCCTTAAC TTC [ ATG ] GGATAT GTAATTGGAAGTTcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 81) and

AATTTCTCGCGAGGTTTCGGcctacAGGGAATTGAAG [TAC] CCTATACATTAACCTTCAAgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 82).

The products amplified by primers 7 and 8 into the template are identical to the products amplified by primers 29 and 30, 31 and 32, 33 and 34, 35 and 36 into the template.

The region indicated by the lowercase letter in the sequence generated by the PCR is a restriction enzyme recognition sequence, the underlined portion is the sequence of the fragment generated by cleavage by restriction enzyme, the site indicated by brackets ([]) is the 'mutation sequence' to be.

The production principle of the amplification product is the same as in Example 1.

2. Cleavage, Purification by Restriction Enzymes and Desalination

It is carried out in the same manner as in Example 1.

3. Maldi- Sat F Mass Spectrometry

It is carried out in the same manner as in Example 1.

rtI169T  (9 mer ) amino acid Codon In 5'-3 'mold
Amplified
Mass of the intercept (Da) In 3'-5 'mold
Amplified
Mass of the intercept (Da) Ile A T T 2802.8 2817.8 Ile A T C 2802.8 2833.8 Ile A T A 2802.8 2808.8 Thr A C T 2787.8 2833.8 Thr A C C 2787.8 2849.8 Thr A C A 2787.8 2824.8 Thr A C G 2787.8 2809.8

rtT184S / A / I / L / F / G / M / C (10 mer ) amino acid Codon Amplified in 5'-3 'template
Mass of the intercept (Da) Amplified from 3'-5 'template
Mass of the intercept (Da) Thr A C T 3058.0 3100.0 Thr A C C 3058.0 3116.0 Thr A C A 3058.0 3091.0 Thr A C G 3058.0 3076.0 Gly G G T 3114.0 3045.0 Gly G G C 3114.0 3061.0 Gly G G A 3114.0 3036.0 Gly G G G 3114.0 3021.0 Ser A G T 3098.0 3060.0 Ser A G C 3098.0 3076.0 Ser T C T 3049.0 3109.0 Ser T C C 3049.0 3125.0 Ser T C A 3049.0 3100.0 Ser T C G 3049.0 3085.0 Cys T G T 3089.0 3069.0 Cys T G C 3089.0 3085.0 Ala G C T 3074.0 3085.0 Ala G C C 3074.0 3101.0 Ala G C A 3074.0 3076.0 Ala G C G 3074.0 3061.0 Ile A T T 3073.0 3084.0 Ile A T C 3073.0 3100.0 Ile A T A 3073.0 3075.0 Leu T T A 3064.0 3084.0 Leu T T G 3064.0 3069.0 Leu C T T 3049.0 3109.0 Leu C T C 3049.0 3125.0 Leu C T A 3049.0 3100.0 Leu C T G 3049.0 3085.0 Phe T T T 3064.0 3093.0 Phe T T C 3064.0 3109.0 Met A T G 3073.0 3060.0

rtS202G / I / C (13 mer ) amino acid Codon Amplified in 5'-3 'template
Mass of the intercept (Da) Amplified from 3'-5 'template
Mass of the intercept (Da) Gly G G A 4093.6 4012.6 Gly G G C 4069.6 4037.6 Gly G G G 4109.6 3997.6 Gly G G T 4084.6 4021.6 Ile A T A 4052.6 4012.6 Ile A T C 4028.6 4037.6 Ile A T T 4043.6 4021.6 Ser A G C 4053.6 4037.6 Ser A G T 4068.6 4021.6 Ala G C T 4027.6 4056.6 Ala G C C 4027.6 4072.6 Ala G C A 4027.6 4047.6 Ala G C G 4027.6 4032.6 Cys T G T 4042.6 4040.6 Cys T G C 4042.6 4056.6

rtIM250V / I / L (12 mer ) amino acid Codon Amplified in 5'-3 'template
Mass of the intercept (Da) Amplified from 3'-5 'template
Mass of the intercept (Da) Met A T G 3755.4 3644.4 Val G T A 3755.4 3659.4 Val G T T 3746.4 3668.4 Val G T C 3731.4 3684.4 Val G T G 3771.4 3644.4 Leu C T G 3731.4 3644.4 Leu C T A 3715.4 3659.4 Leu C T T 3706.4 3668.4 Leu C T C 3691.4 3684.4 Leu T T G 3746.4 3644.4 Leu T T A 3730.4 3659.4 Ile A T T 3730.4 3668.4 Ile A T C 3715.4 3684.4 Ile A T A 3739.4 3659.4

The results of analyzing the size of the fragments produced by the reaction by Maldi-Top Mass Spectrometry are as shown in Tables 4 to 7, and the hepatitis B virus DNA polymerization based on Tables 4 to 7 above. Site-specific mutations in the enzyme's resistance to entacarvir can be determined. If genotypes are mixed, all molecular weights of the base are shown.

As shown in Figure 6 and 7, using the analysis method according to the present invention can determine whether the base mutation of five different regions on the HBV genome as a single diagnostic analysis results.

Example 5 . Nucleotide Variation in Hepatitis B Virus DNA Polymerase Resistant to Adefovir

Nucleotide mutations appear in the DNA polymerase gene of hepatitis B virus that causes hepatitis B in humans. Base mutations in some of these sites cause resistance to adefovir, a therapeutic agent for hepatitis B, and amino acids A181V / T, N236T, and the like have been reported as causative mutations.

One. PCR  Amplification

The sequence (5 '→ 3') of the template DNA to be analyzed is as follows. In Example 5, two base mutations are simultaneously detected. Since the two base mutation sites are separated from each other on the HBV genome, sequence information is described as follows. Each of the two base mutations refers to a variation of the 181 th and 236 th codons, which in turn encodes an HBV polymerase amino acid.

CATCATCCTGGGCTTTCGCAAGATTCCTATGGGAGTGGGCCTCAG TCCGTTTCTC ATGgct CAGTTTACTAGTGCC ATTTGTTCAGTGGTTCGCAGGGCTTTCCCCCACTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAACATCTTGAGTCCCTTTTTACCTCTATTACCAATTTTCTTC TGTCTTTGGGTATACATTT Aaac CCTAATAAAACC AAGCGTTGGGGCTACTCCCTTA (sequence information 83)

Underlined sequences are the sites where primers 37 and 38 and 39 and 40 bind in primer pairs. Bases written in lowercase are 'sequence sequences'.

Primer 37. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTCCGTTTCTC -3' (35mer) (SEQ ID NO: 84)

Primer 38. 5 '-GACGACGCCTCTCCTTTCCTggatgGGCACTAGTAAACTG -3' (40mer) (SEQ ID NO: 85)

Primer 39. 5 '-TTAAAGAGCGCTCCAAAGCCggatgTGTCTTTGGGTATACATTT -3' (44mer) (SEQ ID NO: 86)

Primer 40. 5 '-GACGACGCCTCTCCTTTCCTggatgGGTTTTATTAGG -3' (37mer) (SEQ ID NO: 87)

The primers 37 to 40 are MSPs, primers 37 and 39 are forward primers, and primers 38 and 40 are reverse primers. As described above, primers 37 to 40, which are MSPs, include an external sequence, a restriction enzyme recognition sequence, and a primer binding sequence, respectively. Lowercase sequences are the recognition sequences of Fok1.

In the experimental method including the PCR reaction, 0.1 μM of primers 37 and 38, and 39 and 40 were respectively added, and 0.4 μM of primers 7 and 8, respectively, and the other reaction conditions were the same as in Example 1. However, only PCR reaction is performed under the following conditions.

5 minutes at 94 ° C,

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

94 ℃ 15 seconds 62 ℃ 15 seconds 72 ℃ 15 seconds (35 cycles),

72 ℃ 5 minutes

DNA is extracted from the serum and purified purely according to conventional methods, and DNA can be separated from the serum using the QIAamp DNA Mini Kit 250 (Qiagen 51106). However, the amount to be eluted is determined separately from 40 to 100 μl, and the obtained DNA is not concentrated.

The sequence of fragments generated by PCR is as follows (5 '-> 3').

The products amplified by primers 37 and 38 against the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTCCGTTTCT CCTG [GCT] CA GTTTACTAGTGCCcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 88) and

AATTTCTCGCGAGGTTTCGGcctacAGGCAAAGAGGAC [CGA] GTCAAA TGATCACGG

gtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 89),

The product amplified by the primers 39 and 40 to the template

TTAAAGAGCGCTCCAAAGCCAGGACggatgTGTCTTTGG GTATACATTTA [AA T] CCTAATAAAACCcatccTATCTAGGAAAGGAGAGGCGTCGTC (SEQ ID NO: 90) and

AATTTCTCGCGAGGTTTCGGcctacACAGAAACCCATA TGTAAAT [ TTA ] GGA TTATTTTGGgtaggTCCTTTCCTCTCCGCAGCAG (SEQ ID NO: 91).

The products amplified by primers 7 and 8 into the template are the same as the products amplified by primers 37 and 38 and 39 and 40 into the template.

The region indicated by the lowercase letter in the sequence generated by the PCR is a restriction enzyme recognition sequence, the underlined portion is the sequence of the fragment generated by cleavage by restriction enzyme, the site indicated by brackets ([]) is the 'mutation sequence' to be.

The production principle of the amplification product is the same as in Example 1.

2. Cleavage, Purification by Restriction Enzymes and Desalination

It is carried out in the same manner as in Example 1.

3. Maldi-Top Mass Spectrometry

It is carried out in the same manner as in Example 1.

L180M , A181V / T (9 mer ) amino acid Codon180 Codon181 In 5'-3 'mould
Amplified
Mass of the intercept (Da) In 3'-5 'mould
Amplified
Mass of the intercept (Da) Leu-ala C T G G C T 2754.8 2811.8 Leu-ala C T A G C T 2738.8 2811.8 Leu-ala C T T G C T 2729.8 2811.8 Leu-ala C T C G C T 2714.8 2811.8 Leu-ala T T G G C T 2769.8 2811.8 Met-ala A T G G C T 2778.8 2811.8 Leu-Thr C T G A C T 2738.8 2826.8 Leu-Thr C T A A C T 2722.8 2826.8 Leu-Thr C T T A C T 2713.8 2826.8 Leu-Thr C T C A C T 2698.8 2826.8 Leu-Thr T T G A C T 2753.8 2826.8 Met-thr A T G A C T 2762.8 2826.8 Leu-val C T G G T T 2769.8 2795.8 Leu-val C T A G T T 2753.8 2795.8 Leu-val C T T G T T 2744.8 2795.8 Leu-val C T C G T T 2729.8 2795.8 Leu-val T T G G T T 2784.8 2795.8 Met-val A T G G T T 2793.8 2795.8 Leu-ser C T G T C T 2729.8 2835.8 Leu-ser C T A T C T 2713.8 2835.8 Leu-ser C T T T C T 2704.8 2835.8 Leu-ser C T C T C T 2689.8 2835.8 Leu-ser T T G T C T 2744.8 2835.8 Met-ser A T G T C T 2753.8 2835.8

N236T  (13 mer ) amino acid Codon236 Amplified in 5'-3 'template
Mass of the intercept (Da) Amplified in 3'-5 'template
Mass of the intercept (Da) Asn A A T 4036.6 4092.6 Asn A A C 4036.6 4108.6 Thr A C T 4012.6 4117.6 Thr A C C 4012.6 4133.6 Asn A A T 4052.6 4077.6 Asn A A C 4052.6 4093.6 Thr A C T 4028.6 4102.6 Thr A C C 4028.6 4118.6

The size of the fragments produced by the reaction was analyzed by Maldi-Top Mass Spectrometry, and the results are shown in Tables 8 and 9, and the hepatitis B virus DNA polymerization based on the Tables 8 and 9 was performed. Site-specific mutations in the enzyme's resistance to adefovir can be determined. If genotypes are mixed, all molecular weights of the base are shown. Unlike the conventional techniques, according to the present invention, the 9mer fragment after restriction enzyme treatment of a PCR amplification product has the advantage of codon 181 as well as variation of codon 180 as shown in Table 8 above. . Codon 180 is less directly related to drug resistance of adefovir, but is a region of resistance mutation of other primary therapeutic agents of HBV. Therefore, the present invention confirmed that not only several base mutations separated from each other by more than 32 bases, but also several base mutations included in the fragment 32mer and separated from each other can also be analyzed.

As shown in Figure 8 and 9, using the analysis method according to the present invention can determine whether the base mutation of two different sites on the HBV genome as a single diagnostic analysis results.

1 is a schematic diagram of a PCR amplification process generated from FP and MSP.

FIG. 2 is a Maldi-Top Mass Spectrometry plot of the 35th, 167th and 235th bases of the human GJB2 gene in normal (G / G, T / T, G / G).

3 shows the maldi-tope mass spectrome when the 35th and 167th bases of the human GJB2 gene are normal (G / G, T / T, G / G) and the 235th base is hetero (G /-). Tree illustration.

4 shows that all of the human PDS2 genes are normal, 2162 base C, 2168 base A, IVS7-2 base A, IVS9 + 3 base A, 439 base A, and 365 base. Maldi-tope mass spectrometry plot when the 367th base is C.

5 is a homotype with a 171th base of human FUT2 A, a 960th base of FUT2 A, a base of FY-NULL (rs2814778) A, a base C of TYP-191 (rs1042602), and MID575 (rs140864) Figure of hetero type Maldi-tope mass spectrometry showing both TTC + and TTC deletion at base.

FIG. 6 shows that the 169th codon of HBV polymerase that causes entacavir resistance in nucleotide variation is amino acid isoleucine (Ile) as ATT, the 184th codon is amino acid threonine (Thr) as ACT, and the 202th codon is AGT. Maldi-tope mass spectrometry plot when the genotype of HBV is wild type, with amino acid serine (Ser) and the 250th codon encoding amino acid methionine (Met) with ATG.

Figure 7 shows that the 169th codon of HBV polymerase to induce entacavir resistance during nucleotide shift is amino acid isoleucine (Ile) as ATT, the 184th codon is amino acid threonine (Thr) as ACT, the 202th codon is GGT Maldi-tope mass spectrometry plot when the genotype of HBV is mutant, with the amino acid glycine (Gly) and the 250th codon encoding the amino acid methionine (Met) with ATG.

FIG. 8 shows the 181th codon of HBV virus polymerase which causes adefovir resistance upon base mutation, amino acid alanine (Ala) with GCT, amino acid asparagine (Asn) with AAT, and 180th codon with CTG Maldi-tope mass spectrometry plot of HBV genotype wild-type, encoding amino acid leucine (Leu).

Figure 9 shows that the 181th codon of HBV virus polymerase to induce adefovir resistance at base mutation is amino acid alanine (Ala) with GCT, the 236th codon is amino acid asparagine (Asn) with ACT, and the 180th codon is CTG. Maldi-tope mass spectrometry plot when the genotype of HBV is mutated, encoding the amino acid leucine (Leu).

<110> YOO Wang Don; GeneMatrix Inc. <120> Method for detecting multiple base mutations <160> 91 <170> KopatentIn 1.71 <210> 1 <211> 270 <212> DNA <213> Homo sapiens <400> 1 gattggggca cgctgcagac gatcctgggg ggtgtgaaca aacactccac cagcattgga 60 aagatctggc tcaccgtcct cttcattttt cgcattatga tcctcgttgt ggctgcaaag 120 gaggtgtggg gagatgagca ggccgacttt gtctgcaaca ccctgcagcc aggctgcaag 180 aacgtgtgct acgatcacta cttccccatc tcccacatcc ggctatgggc cctgcagctg 240 atcttcgtgt ccacgccagc gctcctagtg 270 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for GJB2 <400> 2 ttaaagagcg ctccaaagcc ggatggcaga cgatcctggg g 41 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for GJB2 <400> 3 gacgacgcct ctcctttcct ggatgtggag tgtttgttca c 41 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for GJB2 <400> 4 ttaaagagcg ctccaaagcc ggatgttgtc tgcaacacc 39 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for GJB2 <400> 5 gacgacgcct ctcctttcct ggatgttctt gcagcctggc tg 42 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for GJB2 <400> 6 ttaaagagcg ctccaaagcc ggatgatccg gctatgggc 39 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for GJB2 <400> 7 gacgacgcct ctcctttcct ggatgacgaa gatcagctgc 40 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of foreign primers <400> 8 ttaaagagcg ctccaaagcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> Reverse primer of foreign primers <400> 9 gacgacgcct ctcctttcct 20 <210> 10 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 10 ttaaagagcg ctccaaagcc ggatggcaga cgatcctggg gggtgtgaac aaacactcca 60 catccaggaa aggagaggcg tcgtc 85 <210> 11 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 11 aatttctcgc gaggtttcgg cctaccgtct gctaggaccc cccacacttg tttgtgaggt 60 gtaggtcctt tcctctccgc agcag 85 <210> 12 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 12 ttaaagagcg ctccaaagcc ggatgttgtc tgcaacaccc tgcagccagg ctgcaagaac 60 atccaggaaa ggagaggcgt cgtc 84 <210> 13 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 13 aatttctcgc gaggtttcgg cctacaacag acgttgtggg acgtcggtcc gacgttcttg 60 taggtccttt cctctccgca gcag 84 <210> 14 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 14 ttaaagagcg ctccaaagcc ggatgtccgg ctatgggccc tgcagctgat cttcgtcatc 60 caggaaagga gaggcgtcgt c 81 <210> 15 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 15 aatttctcgc gaggtttcgg cctacaggcc gatacccggg acgtcgacta gaagcagtag 60 gtcctttcct ctccgcagca g 81 <210> 16 <211> 50 <212> DNA <213> Homo sapiens <400> 16 aggacacatt ctttttgacg gtccatgatg ctatactcta tctacagaac 50 <210> 17 <211> 45 <212> DNA <213> Homo sapiens <400> 17 acatcttttg ttttatttca gacgataatt gctactgcca tttca 45 <210> 18 <211> 45 <212> DNA <213> Homo sapiens <400> 18 ccatcgatgg gaaccaggta tgggtgccct tttgctgaac tggtt 45 <210> 19 <211> 45 <212> DNA <213> Homo sapiens <400> 19 ttttccagtg gtgagtttaa tggtgggatc tgttgttctg agcat 45 <210> 20 <211> 44 <212> DNA <213> Homo sapiens <400> 20 gtctctactc tgcttttttc cctatcctga catactttat cttt 44 <210> 21 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for PDS2 <400> 21 ttaaagagcg ctccaaagcc aggacggatg cattcttttt ga 42 <210> 22 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for PDS2 <400> 22 gacgacgcct ctcctttcct agataggatg agtatagcat ca 42 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for PDS2 <400> 23 ttaaagagcg ctccaaagcc ggatgttttg ttttatttc 39 <210> 24 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for PDS2 <400> 24 gacgacgcct ctcctttcct ggatggcagt agcaattat 39 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for PDS2 <400> 25 ttaaagagcg ctccaaagcc ggatgcgatg ggaaccaggt 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for PDS2 <400> 26 gacgacgcct ctcctttcct ggatgtcagc aaaagggcac 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for PDS2 <400> 27 ttaaagagcg ctccaaagcc ggatgccagt ggtgagttta 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for PDS2 <400> 28 gacgacgcct ctcctttcct ggatgacaac agatcccacc 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for PDS2 <400> 29 ttaaagagcg ctccaaagcc ggatgtctac tctgcttttt 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for PDS2 <400> 30 gacgacgcct ctcctttcct ggatgaaagt atgtcaggat 40 <210> 31 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 31 ttaaagagcg ctccaaagcc aggacggatg cattcttttt gayggtccrt gatgctatac 60 tcatcctatc taggaaagga gaggcgtcgt c 91 <210> 32 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 32 aatttctcgc gaggtttcgg cctacgtaag aaaaactrcc aggyactacg atatgagtag 60 gtcctttcct ctccgcagca g 81 <210> 33 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 33 ttaaagagcg ctccaaagcc aggacggatg ttttgtttta tttcrgacga taattgctac 60 tgccatccta tctaggaaag gagaggcgtc gtc 93 <210> 34 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 34 aatttctcgc gaggtttcgg cctacaaaac aaaataaagy ctgctattaa cgatgacggt 60 aggtcctttc ctctccgcag cag 83 <210> 35 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 35 ttaaagagcg ctccaaagcc aggacggatg cgatgggaac caggtrtggg tgcccttttg 60 ctgacatcct atctaggaaa ggagaggcgt cgtc 94 <210> 36 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 36 aatttctcgc gaggtttcgg cctacgctac ccttggtcca yacccacggg aaaacgactg 60 taggtccttt cctctccgca gcag 84 <210> 37 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 37 ttaaagagcg ctccaaagcc aggacggatg ccagtggtga gtttartggt gggatctgtt 60 gtcatcctat ctaggaaagg agaggcgtcg tc 92 <210> 38 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 38 aatttctcgc gaggtttcgg cctacggtca ccactcaaat yaccacccta gacaacagta 60 ggtcctttcc tctccgcagc ag 82 <210> 39 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 39 ttaaagagcg ctccaaagcc aggacggatg tctactctgc tttttttccy tatcctgaca 60 tactttcatc ctatctagga aaggagaggc gtcgtc 96 <210> 40 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 40 aatttctcgc gaggtttcgg cctacagatg agacgaaaaa aagggaatag gactgtatga 60 aagtaggtcc tttcctctcc gcagcag 87 <210> 41 <211> 31 <212> DNA <213> Homo sapiens <400> 41 tggacgatca atgcaatagg ccgcctgggg a 31 <210> 42 <211> 32 <212> DNA <213> Homo sapiens <400> 42 cctgccggag tggacaggga ttgccgcaga cc 32 <210> 43 <211> 33 <212> DNA <213> Homo sapiens <400> 43 atgtccaatc tgctcttcaa gttcaaccaa cct 33 <210> 44 <211> 34 <212> DNA <213> Homo sapiens <400> 44 cgcctgtgct tccaagataa gagccaagga ctaa 34 <210> 45 <211> 35 <212> DNA <213> Homo sapiens <400> 45 tgcactgctt gggggatctg aaatctggag agaca 35 <210> 46 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for FUT2A171G <400> 46 ttaaagagcg ctccaaagcc ggatgtggac gatcaatgc 39 <210> 47 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for FUT2A171G <400> 47 gacgacgcct ctcctttcct ggatgtcccc aggcggccta t 41 <210> 48 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for FUT2A960G <400> 48 ttaaagagcg ctccaaagcc ggatgcctgc cggagtggac 40 <210> 49 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for FUT2A960G <400> 49 gacgacgcct ctcctttcct ggatgggtct gcggcaatcc c 41 <210> 50 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for MID575 (rs140864) <400> 50 ttaaagagcg ctccaaagcc ggatgatgtc caatctgctc 40 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <220> Reverse primer of multiple sequence primers for MID575 (rs140864) <400> 51 gacgacgcct ctcctttcct ggatgaggtt ggttgaactt 40 <210> 52 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for          FY-NULL (rs2814778) <400> 52 ttaaagagcg ctccaaagcc ggatgcgcct gtgcttccaa g 41 <210> 53 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for          FY-NULL (rs2814778) <400> 53 gacgacgcct ctcctttcct ggatgttagt ccttggctct ta 42 <210> 54 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for          TYP-191 (rs1042602) <400> 54 ttaaagagcg ctccaaagcc ggatgtgcac tgcttggggg at 42 <210> 55 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for          TYP-191 (rs1042602) <400> 55 gacgacgcct ctcctttcct ggatgtgtct ctccagattt ca 42 <210> 56 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 56 ttaaagagcg ctccaaagcc aggacggatg tggacgatca atgcratagg ccgcctgggg 60 acatcctatc taggaaagga gaggcgtcgt c 91 <210> 57 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 57 aatttctcgc gaggtttcgg cctacacctg ctagttacgy tatccggcgg acccctgtag 60 gtcctttcct ctccgcagca g 81 <210> 58 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 58 ttaaagagcg ctccaaagcc aggacggatg cctgccggag tggacrggga ttgccgcaga 60 cccatcctat ctaggaaagg agaggcgtcg tc 92 <210> 59 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 59 aatttctcgc gaggtttcgg cctacggacg gcctcacctg kccctaacgg cgtctgggta 60 ggtcctttcc tctccgcagc ag 82 <210> 60 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 60 ttaaagagcg ctccaaagcc aggacggatg atgtccaatc tgctcttcaa gttcaaccaa 60 cctcatccta tctaggaaag gagaggcgtc gtc 93 <210> 61 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 61 aatttctcgc gaggtttcgg cctacgacag gttagacgag aagttcaagt tggttggagt 60 aggtcctttc ctctccgcag cag 83 <210> 62 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 62 ttaaagagcg ctccaaagcc aggacggatg cgcctgtgct tccaagrtaa gagccaagga 60 ctaacatcct atctaggaaa ggagaggcgt cgtc 94 <210> 63 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 63 aatttctcgc gaggtttcgg cctacgcgga cacgaaggtt cyattctcgg ttcctgattg 60 taggtccttt cctctccgca gcag 84 <210> 64 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 64 ttaaagagcg ctccaaagcc aggacggatg tgcactgctt gggggatmtg aaatctggag 60 agacacatcc tatctaggaa aggagaggcg tcgtc 95 <210> 65 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 65 aatttctcgc gaggtttcgg cctacacgtg acgaaccccc takactttag acctctctgt 60 gtaggtcctt tcctctccgc agcag 85 <210> 66 <211> 318 <212> DNA <213> Homo sapiens <400> 66 actgcacttg tattcccatc ccatcatcct gggctttcgc aagattccta tgggagtggg 60 cctcagtccg tttctcatgg ctcagtttac tagtgccatt tgttcagtgg ttcgcagggc 120 tttcccccac tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt 180 acaacatctt gagtcccttt ttacctctat taccaatttt cttctgtctt tgggtataca 240 tttaaaccct aataaaacca agcgttgggg ctactccctt aacttcatgg gatatgtttg 300 acccctaata aaaccaaa 318 <210> 67 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 67 ttaaagagcg ctccaaagcc ggatgcctgg gctttcgcaa g 41 <210> 68 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 68 gacgacgcct ctcctttcct ggatgcactc ccatagg 37 <210> 69 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 69 ttaaagagcg ctccaaagcc ggatgttctc ctggctcagt tt 42 <210> 70 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 70 gacgacgcct ctcctttcct ggatgacaaa tggcact 37 <210> 71 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 71 ttaaagagcg ctccaaagcc ggatggtttg gctttc 36 <210> 72 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 72 gacgacgcct ctcctttcct ggatgcaata ccacatgatc catata 46 <210> 73 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 73 ttaaagagcg ctccaaagcc ggatgtccct taacttc 37 <210> 74 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 74 gacgacgcct ctcctttcct ggatgaactt ccaattacat atcc 44 <210> 75 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 75 ttaaagagcg ctccaaagcc aggacggatg cctgggcttt cgcaagattc ctatgggagt 60 gcatcctatc taggaaagga gaggcgtcgt c 91 <210> 76 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 76 aatttctcgc gaggtttcgg cctacggacc cgaaagcgtt ctaaggatac cctcacgtag 60 gtcctttcct ctccgcagca g 81 <210> 77 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 77 ttaaagagcg ctccaaagcc aggacggatg ttctcctggc tcagtttact agtgccattt 60 gtcatcctat ctaggaaagg agaggcgtcg tc 92 <210> 78 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 78 aatttctcgc gaggtttcgg cctacagagg accgagtcaa atgatcacgg taaacagtag 60 gtcctttcct ctccgcagca g 81 <210> 79 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 79 ttaaagagcg ctccaaagcc aggacggatg gtttggcttt cggatatatg gatcatgtgg 60 tattgcatcc tatctaggaa aggagaggcg tcgtc 95 <210> 80 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 80 aatttctcgc gaggtttcgg cctaccaaac cgaaagccta tatacctagt acaccataac 60 gtaggtcctt tcctctccgc agcag 85 <210> 81 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 81 ttaaagagcg ctccaaagcc aggacggatg tcccttaact tcatgggata tgtaattgga 60 agttcatcct atctaggaaa ggagaggcgt cgtc 94 <210> 82 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 82 aatttctcgc gaggtttcgg cctacaggga attgaagtac cctatacatt aaccttcaag 60 taggtccttt cctctccgca gcag 84 <210> 83 <211> 260 <212> DNA <213> Homo sapiens <400> 83 catcatcctg ggctttcgca agattcctat gggagtgggc ctcagtccgt ttctcatggc 60 tcagtttact agtgccattt gttcagtggt tcgcagggct ttcccccact gtttggcttt 120 cagttatatg gatgatgtgg tattgggggc caagtctgta caacatcttg agtccctttt 180 tacctctatt accaattttc ttctgtcttt gggtatacat ttaaacccta ataaaaccaa 240 gcgttggggc tactccctta 260 <210> 84 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 84 ttaaagagcg ctccaaagcc ggatgtccgt ttctc 35 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 85 gacgacgcct ctcctttcct ggatgggcac tagtaaactg 40 <210> 86 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of multiple sequence primers for HBV <400> 86 ttaaagagcg ctccaaagcc ggatgtgtct ttgggtatac attt 44 <210> 87 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of multiple sequence primers for HBV <400> 87 gacgacgcct ctcctttcct ggatgggttt tattagg 37 <210> 88 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 88 ttaaagagcg ctccaaagcc aggacggatg tccgtttctc ctggctcagt ttactagtgc 60 ccatcctatc taggaaagga gaggcgtcgt c 91 <210> 89 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 89 aatttctcgc gaggtttcgg cctacaggca aagaggaccg agtcaaatga tcacgggtag 60 gtcctttcct ctccgcagca g 81 <210> 90 <211> 95 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 90 ttaaagagcg ctccaaagcc aggacggatg tgtctttggg tatacattta aatcctaata 60 aaacccatcc tatctaggaa aggagaggcg tcgtc 95 <210> 91 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Resulting PCR Fragment <400> 91 aatttctcgc gaggtttcgg cctacacaga aacccatatg taaatttagg attattttgg 60 gtaggtcctt tcctctccgc agcag 85  

Claims (14)

a) amplifying a specific polynucleotide using multiple sequence primers (MSP) and external primers (FP); b) cutting the amplified specific polynucleotides with restriction enzymes to generate two or more single-stranded polynucleotide fragments containing a mutation sequence having a size of 2 to 32 bases; And c) multi-nucleotide variation analysis method comprising the step of measuring the molecular weight of the cleaved section. The method of claim 1, In the step a), the multiple sequence primers are used in pairs of several base variants, each separated by more than 32 bases present in a specific polynucleotide, and the outer primers are used in pairs. Multiple base mutation analysis method. The method of claim 2, The multiple sequence primer pair consists of a forward primer and a reverse primer, and the forward primer includes an external sequence 1, a restriction enzyme recognition sequence and a primer binding sequence 1, and the reverse primer is an external sequence 2, a restriction enzyme recognition sequence and a primer Involving sequence 2 Multiple base mutation analysis method. The method of claim 3, The forward primer is selected from the group consisting of primers of SEQ ID NOs: 2, 4, 6, 21, 23, 25, 27, 29, 46, 48, 50, 52, 54, 67, 69, 71, 73, 84, and 86 One or more Multiple base mutation analysis method. The method of claim 2, The outer primer pair is composed of a forward primer and a reverse primer, wherein the forward primer includes outer sequence 1, and the reverse primer includes outer sequence 2 Multiple base mutation analysis method. The method of claim 5, The forward primer is a primer of SEQ ID NO: 8 Multiple base mutation analysis method. The method according to claim 3 or 5, The external sequence 1 and the external sequence 2 are the same or different, and the primer binding sequence 1 and primer binding sequence 2 are the same or different. Multiple base mutation analysis method. The method of claim 1, Adjusting the annealing temperature so that the reaction of the outer primer does not proceed during the reaction of the multi-sequence primer in step a), and adjusts the annealing temperature so that the outer primer reacts after the multi-stage primer is exhausted. Multiple base mutation analysis method. The method of claim 8, The amount of the multiple sequence primer is added to 0.03 μM to 0.15 μM Multiple base mutation analysis method. The method of claim 8, PCR reaction of the multiple sequence primer 2 to 15 times Multiple base mutation analysis method. The method of claim 8, The annealing temperature at which the reaction of the multiple sequence primer proceeds is different from the annealing temperature at which the outer primer reacts. Multiple base mutation analysis method. The method of claim 8, The annealing temperature at which the reaction of the multiple sequence primer proceeds is 40 ° C. to 55 ° C., and the annealing temperature at which the external primer is reacted is 60 ° C. to 70 ° C. Multiple base mutation analysis method. The method of claim 1, The specific polynucleotide of step a) may be used for several different base mutation sites of GJB2, several different base mutation sites of PDS2, several different SNP marker sites, and entacarvir of hepatitis B virus DNA polymerase. Several different base mutation sites related to resistance, and any one selected from the group consisting of several different base mutation sites related to adefovir resistance of hepatitis B virus DNA polymerase. Multiple base mutation analysis method. The method of claim 13, The SNP marker includes one or more selected from the group consisting of FUT2A171G, FUT2A960G, MID575 (rs140864), FY-NULL (rs2814778), and TYP-191 (rs1042602). Multiple base mutation analysis method.

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