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Kit for detecting polymorphic site genetic typing of key enzyme genes of folic acid metabolism and detection method thereof

A kit and genotyping technology, applied in the fields of biological science and clinical medical diagnosis, can solve problems such as application limitations, high sample quality requirements, and difficult typing of reversed mutation sites, so as to achieve the effect of avoiding pollution and precise pollution

Active Publication Date: 2017-07-18
上海蕴亲基因科技有限公司
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Problems solved by technology

However, due to its high requirements on sample quality, it is difficult to type some inverted mutation sites and is limited in application.

Method used

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  • Kit for detecting polymorphic site genetic typing of key enzyme genes of folic acid metabolism and detection method thereof
  • Kit for detecting polymorphic site genetic typing of key enzyme genes of folic acid metabolism and detection method thereof
  • Kit for detecting polymorphic site genetic typing of key enzyme genes of folic acid metabolism and detection method thereof

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Embodiment Construction

[0051] The above scheme will be further explained below in conjunction with specific embodiments

[0052] 1. Experimental material processing:

[0053] Put the wiped oral sample cotton swab in a 0.5ml centrifuge tube, use scissors to cut the cotton swab part from the stem, add 300 μl of extraction-free solution, vortex for 10 seconds to mix. Lyse cells at 65°C for 30 minutes.

[0054] 2. Probe hybridization melting curve method for genotyping

[0055] a. The genotyping detection system for key enzymes of folate metabolism is designed mainly for MTHFR and MTRR genes. Based on the probe hybridization melting curve genotyping method, the two SNP loci (rs1801131 and rs1801133) on the MTHFR coding gene and the SNP rs1801394 on the MTRR coding gene were genotyped. According to the distribution of these three mutation sites in the corresponding gene sequence, we first designed three fluorescent probes covering these mutation sites, and then designed three pairs of specific amplification ta...

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Abstract

The invention provides a method for typing three sites of key enzyme genes (MTHFR and MTRR) for folic acid metabolism by utilizing a fluorescence labeling probe and combining with a multiple PCR method. An amplimer and a fluorescent probe are designed according to MTHFR and MTRR genes; three sections of DNA sequences are detected in the amplification of a PCR amplifier; fluorescence signals which are released in amplification and dissolution processes in a reaction system are detected; a result is judged in the manner of (1) judging a typing result according to a Tm value shown by a hybrid peak of wild type and mutant type standard plasmids and (2) simultaneously typing according to the magnitude of a fluorescence value of an amplification curve. The primer and the probe adopted by the invention are high in specificity and sensitivity; the detection for three sites is simultaneously performed; the operation is simple and the result is easily judged; the typing result is more accurate and reliable in the manner of twice calibrating typing. The method provided by the invention can be applied to the aspects of guiding the pregnant woman to orally take and supplement folic acid, prompting the high risk in cerebral apoplexy, coronary heart disease and venous thrombus, assessing the metabolic activity of folic acid, and the like.

Description

Technical field [0001] The invention belongs to the field of biological science and clinical medical diagnosis, and relates to a method for detecting polymorphic sites of key enzyme genes in folate metabolism by using a multiple fluorescent PCR method. Using the method of the present invention, the simultaneous typing and detection of multiple SNP sites can be completed at one time, with strong specificity, high sensitivity, and simple operation. It can be used to guide pregnant women to orally supplement the dose of folic acid, prompting stroke, coronary heart disease and venous thrombosis. High-risk and folic acid metabolic activity assessment and other aspects are applied. Background technique [0002] Methylenete-trahydmfolate reductase (methylenete-trahydmfolate reductase, MTHFR) is a key enzyme in the process of folate metabolism, which can convert reduced folate into 5-methyltetrahydrofolate (5-MTHF), the former being thymidylic acid One of the important raw materials for...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 熊乾斌
Owner 上海蕴亲基因科技有限公司
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