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Method for detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism by using high-resolution melting curve analysis technology

A gene polymorphism, high-resolution melting technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of primer design limitations, unsuitable detection, high cost, etc., and achieve fast detection speed and low cost. Low, easy-to-operate effect

Inactive Publication Date: 2014-01-22
上海中优医药高科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has few steps, is not easy to pollute, and is convenient for typing large samples; however, it has certain restrictions on primer design and certain requirements on the sequence near the SNP, which is limited by the position and type of the mutated base
Direct DNA sequencing can directly provide accurate base information and is regarded as the gold standard for mutation detection, but it is time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Method for detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism by using high-resolution melting curve analysis technology
  • Method for detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism by using high-resolution melting curve analysis technology
  • Method for detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism by using high-resolution melting curve analysis technology

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Experimental program
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Embodiment

[0020] 1. The genomic DNA of oral epithelial cells of the subject is extracted by silica gel adsorption method, and the concentration and purity of DNA are detected by electrophoresis gel imaging method. The concentration of the sample to be tested is standardized to 10ng / ul;

[0021] The negative control DNA is deemed qualified when it meets the following conditions: OD value A260 / A280 is between 1.8-2.0; electrophoresis band is clear; sequencing identified MTHFR C677T locus genotype as CC;

[0022] Positive control 1 DNA is deemed qualified when it meets the following conditions: OD value A260 / A280 between 1.8-2.0; electrophoresis band is clear; sequencing identified MTHFR C677T locus genotype as CT;

[0023] Positive control 2 DNA is deemed qualified when it meets the following conditions: OD value A260 / A280 between 1.8-2.0; electrophoresis band is clear; sequencing identified MTHFRC677T locus genotype as TT.

[0024] 2. According to the conservative region of the MTHFR gene, use th...

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Abstract

The invention discloses a method for detecting 5,10-methylene tetrahydrofolate reductase gene typing by using a high-resolution melting curve analysis technology. The method is characterized by comprising the following steps: extracting genome DNA (Deoxyribose Nucleic Acid) of an oral epithelial cell / peripheral blood cell sample of a detected person by adopting a silica gel adsorption method; designing a detection primer containing a target site; carrying out site typing on a PCR (Polymerase Chain Reaction) amplified gene sequence by using the high-resolution melting curve analysis technology. The method disclosed by the invention is high in sensitivity, good in specificity and fast in detection speed, and can be used for predicating the drug susceptibility and the tolerance of methotrexate.

Description

Technical field [0001] The invention relates to a detection method for 5,10-methylenetetrahydrofolate reductase genotyping, and belongs to the technical field of molecular biology. Background technique [0002] 5,10-methylenetetrahydrofolate reductase (MTHFR) is located on chromosome lp36. The full-length cDNA is 2220bp. The gene includes 11 exons and 10 introns. It is 5,10-methylenetetrahydrofolate reductase (NADPH)( Methylenetetrahydrofolate reductase) protein coding gene whose main function is to convert 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate with biological functions in the pathway of folate metabolism. 5-Methyltetrahydrofolate can further enter the methyl transfer pathway and indirectly provide methyl groups for DNA methylation and protein methylation through the re-methylation process of homocysteine ​​and make the blood homotype The amount of cysteine ​​is kept at a low level. MTHFR gene regulates DNA synthesis, activation, repair, etc. Its polymorp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2527/107C12Q2563/107C12Q2600/106C12Q2600/156C12Q2531/113
Inventor 吴颖臻张奕傅咏南王校毛丹丹卞雪莲
Owner 上海中优医药高科技股份有限公司