Method for detecting polymorphism of MTHFR gene through fluorescent quantitative PCR
A technology of gene polymorphism and fluorescence quantification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of amplification product contamination, cumbersome operation steps, and long detection cycle. Achieve the effects of improving detection efficiency, simple operation, and improving specificity
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Embodiment 1
[0031] A method for preparing a DNA sample to be tested in a method for detecting MTHFR gene polymorphisms, comprising the steps of: extracting genomic DNA with a blood / cell / tissue genomic DNA extraction kit (DP304), and using NP80-touch (Germany IMPLEN) Determine the concentration and purity of the DNA, and then save the genomic DNA.
Embodiment 2
[0033] 1. Primer and Probe Design
[0034] The present invention designs specific primers and specific fluorescent probes respectively according to the base sequences of c.677C>T and c.1298A>C sites of the MTHFR gene, and the specific primers are designed at both ends of the SNP sites by Primer Premier 5.0 Primers, select the most suitable primers through software analysis; specific probes are located in the region between a pair of primers, where the Tm value should be between 60-70°C, usually 5-10°C higher than the primers. The 5' end of the probe for detecting the wild type is labeled with a fluorescent reporter group (FAM), the 5' end of the probe for detecting the mutant type is labeled with a fluorescent reporter group (VIC), and the 3' end is labeled with a non-fluorescent quencher group ( NFQ) label, and the MGB modification group is also linked to the probe; in actual operation, the fluorescent reporter group can be replaced according to the actual situation, as long ...
Embodiment 3
[0050] Embodiment 3: the configuration of kit
[0051] According to the above experimental results, the present embodiment provides a preferred kit for the detection of MTHFR gene polymorphisms by fluorescent quantitative PCR, which includes the following reagents:
[0052] 1. 2×TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (including dUTP);
[0053] 2. Probe Mix: two sets of fluorescent probes, the final concentration is 0.6μM;
[0054] 3. Primer Mix: two sets of primer pairs, F upstream primer and R downstream primer have a final concentration of 0.6 μM;
[0055] 4. Sample DNA extraction reagents;
[0056] 5. Sample collection storage box (such as containing oral swabs and swab storage boxes / tubes);
[0057] 6. Ultrapure water.
[0058] The reagents are properly placed in the kit, and then put into the instructions for use of the kit (optionally reconfigure PCR test tubes or orifice plates or pipette guns). The instructions for use include the col...
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