Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe

A gene polymorphism and detection kit technology, applied in the field of genetic testing, can solve the problems of prone to false positives, difficult interpretation of results, troublesome operation, etc., to reduce the risk of false positives, facilitate high-throughput detection, The effect of improving the efficiency of detection

Inactive Publication Date: 2014-04-30
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Advantages: accurate results, sequencing method is the gold standard for gene polymorphism site detection; Disadvantages: high cost, cumbersome operation, long test period
Advantages: low cost, intuitive results; Disadvantages: Some polymorphic sites do not have suitable restriction enzyme sites, and mutations need to be introduced manually. In addition, the operation is cumbersome and the test cycle is long
Disadvantages: PCR products need to be analyzed later, and the operation is cumbersome; in addition, the results are not easy to interpret, and false positives are prone to occur
Disadvantages: A sample needs to be divided into 2 tubes for PCR testing to detect the genotype of a SNP site, which is troublesome to operate
Advantages: One SNP site genotype only needs one tube of PCR to complete the detection; Disadvantages: It is not easy to interpret the difference in Tm value according to the HRM melting curve, and the difference in the peak diagram of the melting curve of different genotypes is not obvious
[0011] At present, the Taqman probe method has the following disadvantages: the results of different genotypes are not significantly different, and three genotype controls must be set up for each qPCR test. In addition, the fluorescence quenching of the TaqMan probe is not complete, and the background is high

Method used

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  • Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe
  • Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe
  • Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: PCR reaction solution configuration in the kit

[0063] Prepare PCR reaction solution for detecting C677T and A286C polymorphism sites of human MTHFR gene respectively. The PCR reaction solution contains primers and corresponding Taqman-MGB probes, Taq enzyme, dNTP mixture, MgCl2 solution, and fluorescent quantitative PCR reaction buffer solution, ddH2O.

[0064] The reaction system for each PCR amplification is 20ul, including 2.0μl of 10×PCR reaction buffer, 2.4μl of 25mMMgCl2 solution, 1.6μl of 2.5mM dNTP mixture, 0.1μl of 5U / ul Taq enzyme, and 1μl of DNA template (about 50ng) , 0.5μl each of 20uM upstream and downstream primers, 0.5μl each of 10uM mutant wild-type probe, ddH2O 12.7μl

[0065] Primers and Taqman-MGB probes in the MTHFR C677T reaction solution:

[0066] Upstream primer MTHFR 677F: 5'-TGTGCTGTGCTGTTGGAAGGTG-3';

[0067] Downstream primer MTHFR 677R: 5'-TCAGAGCCCCCAAAGCAGAGGACTC-3';

[0068] Mutation probe MTHFR 677T: 5'-FAM-TCATGGCATTTCT...

Embodiment 2

[0075] Embodiment 2: the use of detection kit

[0076] 1. Extract DNA template

[0077] For human blood DNA extraction, Genomic DNA was extracted from blood using the Whole Blood DNA Extraction Kit of Tiangen Biochemical Biotechnology Company.

[0078] 2. PCR reaction system configuration

[0079] Use the PCR reaction solution configured in Example 1 for detection, one sample, and 2 tubes of PCR detection at the same time. Take 19ul MTHFR C677T PCR reaction solution into one PCR reaction tube, take 19ul MTHFR A286C PCR reaction solution into another PCR reaction tube, add 1ul (10~ 100ng).

[0080] 3. Fluorescent PCR detection

[0081] Put the configured PCR system into a fluorescent PCR instrument for fluorescent PCR amplification detection; the reaction conditions are: denaturation and enzyme activation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 30 minutes , cycle 45 times.

[0082] Fluorescent...

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Abstract

The invention belongs to the field of genetic gene detection, and in particular relates to a kit and a method for detecting the gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on a Taqman-MGB (Minor Groove Binder) probe. By adopting the kit and method, two SNP (single nucleotide polymorphism) sites namely C677t and A1298C can be detected at the same time, and the results are easy to judge and read. The MGB probe has a nonluminous 3'-end quenching group and a relatively low background, is more sensitive to detection of a single base mutation, and can judge and read different genotypes only according to a Ct value, and the result can be judged and read more easily compared with that of conventional methods of Taqman probe detection and HRM (high resolution melting) detection. The probe price is relatively low, the detection cost is relatively low, a polymorphism site only needs one-tube qPCR (quantitative polymerase chain reaction) detection, and the operation is simple. Moreover, the method disclosed by the invention does not need subsequent analysis of PCR products, so that while the detection cost is saved, the detection circle is greatly shortened, the detection efficiency is improved and the risk of false positive caused by PCR product pollution is reduced.

Description

technical field [0001] The invention belongs to the field of genetic detection, in particular to a human MTHFR gene polymorphism detection kit and method based on Taqman-MGB probes. Background technique [0002] 5,10-methylenetetrahydrofolate reductase (Mthylenetetrahydrofolate reductase, MTHFR) is a key enzyme in the folate metabolism system, catalyzing the reduction of 5,10-methylenetetrahydrofolate to the methyl donor 5-methyltetrahydrofolate folic acid. On the one hand, 5-methyltetrahydrofolate can be used as an indirect donor of methyl groups to participate in the synthesis of purines and pyrimidines and the methylation of DNA, RNA, and proteins; on the other hand, under the catalysis of methionine synthase, Vitamin B12 is used as a coenzyme to re-methylate homocysteine ​​in the blood to produce methionine, thereby maintaining normal homocysteine ​​levels in the body. There are two common polymorphic sites in the MTHFR gene, C677T (SNP ID: rs1801133) and A1296C (SNP I...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨曦
Owner HENAN UNIV OF SCI & TECH
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