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A method and kit for detecting the genotype of the mthfr gene rs1801131 polymorphic site

A technology of polymorphic sites and genotypes, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as misjudgment, achieve accuracy, easy operation, and low detection cost low effect

Active Publication Date: 2022-02-11
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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  • A method and kit for detecting the genotype of the mthfr gene rs1801131 polymorphic site
  • A method and kit for detecting the genotype of the mthfr gene rs1801131 polymorphic site
  • A method and kit for detecting the genotype of the mthfr gene rs1801131 polymorphic site

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Embodiment 1

[0059] This embodiment is designed with a pair of primers, and the upper prismatic 3 'end uses a mismatch base, and the PCR is amplified to form a polymorphic bit recognition sequence GaATM [M = A, C base. The corresponding sequence of the mutant allele C is GAATC, which can be recognized by the restriction nucleic acid endozyme Hinfi; the corresponding sequence of wild-type allele A is Gaata, cannot be recognized by Hinfi], downstream primers are conventional primers, and MTHFR can be Gene's No. 17018 to 17040 Base base sequence reverse complementary. The PCR product comprises a solid Hinfi recognition sequence GACTC as an enzyme-cutting control. The specific scheme is as follows:

[0060] 1. Genomic DNA extraction

[0061] The genomic DNA was extracted by the operation steps of the specification using an oral Wink genomic DNA extraction kit. The source of DNA is not limited thereto, and the DNA from any somatic cell source can be.

[0062] 2. Primer design

[0063] The sequence ...

Embodiment 2

[0098] The present embodiment uses both the upstream primer mismatches formed by PCR amplification of polymorphic site recognition sequences GAATM [M = A, C bases. Sequences corresponding to the mutant allele C GAATC, can be cut restriction endonuclease HinfI recognition; the corresponding sequence of the wild type allele A was GAATA, can not be identified HinfI], but also through the downstream mismatched PCR primers amplification forming a HinfI recognition sequence as the restriction control. The specific scheme is as follows:

[0099] 1. Genomic DNA

[0100] Oral swab using genomic DNA extraction kit, according to the steps of the instructions extracted genomic DNA. DNA source is not limited thereto, any DNA can be derived from somatic cells.

[0101] 2. primer design

[0102] 500bp sequence of each retrieval before and after the MTHFR gene rs1801131 polymorphic site (see figure 1 , image 3 ), Using PrimerPremier 5.0 software design primers specific for the type of site. Where...

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Abstract

The invention discloses a detection MTHFR A method and a kit for genotyping polymorphic sites of gene rs1801131. The present invention detects based on the PCR-RFLP method MTHFR In the process of gene rs1801131 polymorphic site genotype, a low-cost endonuclease was selected, and a unique "internal quality control" mechanism was introduced at the same time, that is, an internal control enzyme cutting site was introduced into the PCR product, which can The incomplete digestion can be identified according to the enzyme digestion map, and the interference of residual PCR products or intermediate products of digestion can be eliminated, and the genotype can be accurately judged according to the characteristic bands, which greatly improves the accuracy of the detection results.

Description

Technical field [0001] The present invention belongs to the field of molecular detection, and more particularly to a method and kit for detecting MTHFR gene RS1801131 polymorphic loci genotypes. Background technique [0002] 5,10-methylene tetrahydrofolate reductase [5, 10-methylenetetrahydrofolate reductase, mthfr] is a critical enzyme in the folic acid metabolism system, and some polymorphisms of the encoding gene can be reduced by enzyme activity, causing folic acid metabolism. A variety of diseases involving (1) maternal health care: neural tube defects, congenital heart disease, lactate, pregnancy hypertension, spontaneous abortion; (2) tumor: lung cancer, gastric cancer, colorectal cancer, etc. (3) Hypertension, coronary heart disease, cerebral infarction, cerebral hemorrhage; (4) mental illness, etc. [0003] MTHFR gene RS1801131 single nucleotide multi-state position (related sequence data see figure 1 The mutation of A → C in the open reading box [A1286C, the old name A1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12N15/11
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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