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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 field of identifying human MTHFR gene polymorphism rs2274976, can solve the problems of difficulty in universal use, expensive endonuclease, and difficulty in selection and identification, and achieves the effect of reducing costs.

Inactive Publication Date: 2014-02-12
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), and primers synthesized by Shanghai Sangon Company.

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

[0088] PCR product sequencing was completed by Shanghai Sangon Bioengineering Co., Ltd.

[0089] 1.2 Sequence search and primer design

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

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

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

[0093] 1.3 Extract DNA from whole blood samples ...

Embodiment 2

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

[0112] The steps in Example 1 are basically the same, 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 endonuclease used It is HpaII (NEB Corporation).

[0113] result:

[0114] The obtained amplification product sequence 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 enzyme digestion, and the sequence is as follows:

[0119] crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60

[0120] gacaactact tcctggtcaa cctggtggac ...

Embodiment 3

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

[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 was MspI (NEB Company).

[0127] DNA extraction:

[0128] Collect 400 μl of human peripheral blood in a common blood collection tube, and use the RelaxGene Rlood DNA System blood genomic DNA extraction system from TIANGEN Company to extract the genomic DNA in the peripheral blood clot as the human genomic DNA template to be tested;

[0129] After the serum in the blood collection tube is separated out, separate the serum, and then follow the steps below:

[0130] Grind the bl...

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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 is December 24, 2009, and the invention title is "Method for Identifying Human MTHFR Gene Polymorphism rs2274976". technical field [0002] The present invention relates to methods of 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 DNA sequence variation caused by the change of a single nucleotide, including single base substitution, insertion and deletion. SNP has been widely used in genetic linkage analysis and association analysis of simple and complex diseases, as well as the location of disease susceptibility genes, and guides the cloning of susceptibility genes. Polymerase chain reaction-restriction fragment length poly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王威姚武杨永利段晓冉王团伟王郁万传君
Owner ZHENGZHOU UNIV
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