Method and kit for detecting rs1801131 polymorphic site genotype in MTHFR (5,10-methylenetetrahydrofolate reductase) gene

A polymorphic site and genotype technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. low effect

Active Publication Date: 2017-08-18
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If an AA-type sample is incompletely digested, with both 147 and 32bp bands and a residual 189bp fragment, it will likely be misjudged as an AC-type sample

Method used

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  • Method and kit for detecting rs1801131 polymorphic site genotype in MTHFR (5,10-methylenetetrahydrofolate reductase) gene
  • Method and kit for detecting rs1801131 polymorphic site genotype in MTHFR (5,10-methylenetetrahydrofolate reductase) gene
  • Method and kit for detecting rs1801131 polymorphic site genotype in MTHFR (5,10-methylenetetrahydrofolate reductase) gene

Examples

Experimental program
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Embodiment 1

[0059] In this embodiment, a pair of primers is designed, and a mismatched base is used at the 3' end of the upstream primer, and the polymorphic site recognition sequence GAATM [M=A, C bases is formed after PCR amplification. The corresponding sequence of the mutant allele C is GAATC, which can be recognized by the restriction endonuclease HinfI; the corresponding sequence of the wild-type allele A is GAATA, which cannot be recognized by HinfI], and the downstream primers are conventional primers, which can be combined with MTHFR The 17018th to 17040th base sequence of the gene is reverse complementary. The PCR product contains an inherent HinfI recognition sequence GACTC used as an internal control for enzyme digestion. The specific plan is as follows:

[0060] 1. Genomic DNA Extraction

[0061] Using the buccal swab genomic DNA extraction kit, the genomic DNA was extracted according to the operation steps in the manual. The DNA source is not limited thereto, and any soma...

Embodiment 2

[0098] In this embodiment, the polymorphic site recognition sequence GAATM [M=A, C bases is formed by PCR amplification using mismatched upstream primers. The corresponding sequence of the mutant allele C is GAATC, which can be recognized by the restriction endonuclease HinfI; the corresponding sequence of the wild-type allele A is GAATA, which cannot be recognized by HinfI]. Amplified to form a HinfI recognition sequence used as an internal control enzyme digestion. The specific plan is as follows:

[0099] 1. Genomic DNA Extraction

[0100] Using the buccal swab genomic DNA extraction kit, the genomic DNA was extracted according to the operation steps in the manual. The DNA source is not limited thereto, and any somatic cell-derived DNA is acceptable.

[0101] 2. Primer Design

[0102] Get the sequence of 500bp before and after the rs1801131 polymorphic site of the MTHFR gene (see figure 1 , image 3 ), using PrimerPremier 5.0 software to design specific typing primers...

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Abstract

The invention discloses a method and a kit for detecting rs1801131 polymorphic site genotype in MTHFR (5,10-methylenetetrahydrofolate reductase) gene. In a process of detecting the rs1801131 polymorphic site genotype in the MTHFR gene on the basis of a PCR-RFLP method, endonuclease, which is low in cost, is adopted; meanwhile, by introducing a specific 'internal quality control' mechanism, namely introducing an internal control restriction enzyme cutting site to a PCR product, the circumstance of incomplete enzyme cutting can be identified in accordance with a restriction map, and interference from residual PCR product or intermediate products of enzyme cutting can be prevented; and the genotype can be accurately judged in accordance with characteristic bands, so that the accuracy of a detection result is greatly enhanced.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a method and a kit for detecting the genotype of the rs1801131 polymorphic site of MTHFR gene. Background technique [0002] 5,10-methylenetetrahydrofolate reductase [5,10-methylenetetrahydrofolate reductase, MTHFR] is a key enzyme in the folate metabolism system, and certain polymorphisms in its coding gene can lead to reduced enzyme activity and cause folate metabolism disorders , thus causing a variety of diseases, involving (1) maternal and child health care: neural tube defects, congenital heart disease, cleft lip and palate, hypertensive disorders during pregnancy, spontaneous abortion; (2) tumors: lung cancer, gastric cancer, colorectal cancer, etc. ; (3) Hypertension, coronary heart disease, cerebral infarction, cerebral hemorrhage; (4) Mental diseases, etc. [0003] MTHFR gene rs1801131 single nucleotide polymorphism site (for related sequence data, see f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2525/131C12Q2521/301
Inventor 何震宇陈杏勇郑裕彤游娟陈嘉鋆颜珍珠苏健南
Owner GUANGDONG PHARMA UNIV
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