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Kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI

A technology of rs2274976 and gene polymorphism, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of difficulty in popularization and use, high price of endonucleases, and impact on costs, and achieve the effect of reducing costs

Inactive Publication Date: 2012-09-19
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the endonuclease BsrBI is often used to identify the human MTHFR gene polymorphism rs2274976. The endonuclease is expensive (refer to the following table 1 for the reference price of some endonucleases), which affects the cost of the experiment and is difficult to test in the laboratory. popular use in
And due to the inherent defects of the traditional PCR-RFLP method, it is usually difficult for those skilled in the art to select other restriction endonucleases to identify the human MTHFR gene polymorphism rs2274976

Method used

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  • Kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI
  • Kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI
  • Kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] 1 Materials and methods

[0085] 1.1 Main reagents and instruments

[0086] Reagents: 2×PCR mix (MBI company), restriction endonuclease MspI (MBI company), agarose (BBI company), primers synthesized by Shanghai Sangon company.

[0087] Instruments: 9600 PCR instrument (PE company), micro electrophoresis tank (Pharmacia Biotech, EPS1000), Gel Doc 2000 gel imager (Bio-RAD company).

[0088] The PCR product sequencing was completed by Shanghai Shenggong Biological Engineering Co., Ltd.

[0089] 1.2 Sequence search and primer design

[0090] Find the MTHFR gene sequence and SNP information on the NCBI website, determine the base variation information of the MTHFR polymorphic site based on relevant literature, and design primers, as follows:

[0091] The forward primer sequence is 5'-CTTTGCCCTGTGGATTGACC-3' (SEQ ID NO:1),

[0092] The reverse primer sequence is 5'-GCAGTTGTCCAGTGGGAAGTCA-3' (SEQ ID NO: 2).

[0093] 1.3 Extract DNA from whole blood samples as DNA to be tested

[0094] EDTA a...

Embodiment 2

[0111] Example 2 Determination of human MTHFR gene rs2274976 polymorphism in whole blood samples from peripheral blood:

[0112] The steps are basically the same as in Example 1, except that the forward primer used in the PCR reaction is SEQ ID NO: 1, the reverse primer is SEQ ID NO: 7, the annealing temperature is 59°C, and the restriction enzyme used is It is HpaII (NEB company).

[0113] result:

[0114] The sequence of the amplified product obtained is as follows (159bp, SEQ ID NO: 8):

[0115] ctttgccctg tggattgacc rgtggggaaa gctgtatgag gaggagtccc cgtcccgcac 60

[0116] catcatccag tacatccacg acaactactt cctggtcaac ctggtggaca atgacttccc 120

[0117] actggacaac tgcctctggc aggtggtgga agacacatt 159

[0118] When the MTHFR polymorphism contains a G allele, a 140bp product (SEQ ID NO: 9) can be formed after restriction digestion. The sequence is as follows:

[0119] crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60

[0120] gacaactact tcctggtcaa cctggtggac aatgacttcc cactg...

Embodiment 3

[0125] Example 3 Determination of the rs2274976 polymorphism of human MTHFR gene in peripheral blood clot samples:

[0126] The steps are basically the same as in Example 1, except that the following method is used to extract DNA from the peripheral blood clot specimen as the DNA to be tested. In addition, the forward primer used in the PCR reaction is SEQ NO: 11 (tttgccctgt ggattgacc), the reverse primer is SEQ NO: 12 (tctcgcattc tgggtggg), and the annealing temperature is 58°C. The restriction endonuclease is MspI (NEB).

[0127] Extract DNA:

[0128] Use ordinary blood collection tube to collect 400μl of human peripheral blood, and use TIANGEN's RelaxGene Rlood DNA System blood genomic DNA extraction system to extract genomic DNA from peripheral blood clots as the test human genomic DNA template;

[0129] After the serum in the blood collection tube is separated, the serum is separated, and then proceed as follows:

[0130] Grind the clot in the blood collection tube into a homogen...

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Abstract

The invention discloses a kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI. The kit comprises a forward primer and a reverse primer for amplifying sequences near a human MTHFR gene polymorphism rs2274976 locus, wherein the last base at the 3'end of the forward primer is adjacent to an rs2274976 polymorphic base, and the last second base of the forward primer is a mismatched base C, so that the forward primer and polymorphic G allele form a CCGG structure in an amplification product; and the forward primer is shown as SEQ ID NO:1 or SEQ NO:11, and the reverse primer is shown as SEQ ID NO:2, SEQ ID NO:7 or SEQ NO:12. The kit also comprises restriction enzyme MspI or HpaII, and an instruction book for implementing identification. The kit is easy to operate, low in cost, and wide in application range.

Description

[0001] This application is a divisional application of the invention patent application with the application number "200910227637.3", the application date on December 24, 2009, and the invention title "Method for Identifying Human MTHFR Gene Polymorphism rs2274976". Technical field [0002] The present invention relates to methods for identifying single nucleotide polymorphisms. More specifically, the present invention relates to a method for identifying the human MTHFR gene polymorphism rs2274976. Background technique [0003] SNP (Single Nucleotide Polymorphism) refers to the variation of DNA sequence caused by a single nucleotide change, including single base substitutions, insertions and deletions. SNP is now widely used in genetic linkage analysis, association analysis, and location of disease susceptibility genes for simple and complex diseases, to guide the cloning of susceptibility genes. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) te...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王威姚武杨永利
Owner ZHENGZHOU UNIV
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