Human MTHFR gene polymorphism detection kit
A detection kit and kit technology, applied in the biological field, can solve the problems of long operation time, unfavorable large-scale promotion, low cost, etc., and achieve the effect of stable typing detection
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Embodiment 1
[0063] 1. Primer and probe synthesis:
[0064] Design and synthesize 2 sets of specific primers SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6; 2 sets of specific probes SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQID NO:10, and label FAM, HEX, Cy3 and Cy5 fluorescent groups at the 5' end, and NFQ-MGB at the 3' end without luminescent quenching Killing group; 2 enrichment primers SEQ ID NO:14, SEQ ID NO:15. The primers and probes were prepared into 100 μM stock solutions for storage.
[0065] 1. Prepare the internal standard system: design and synthesize a pair of internal standard primers designed for the human genome, the primer pair sequences are SEQ ID NO: 11 and SEQ ID NO: 12; design and synthesize internal standard probes, the probes is SEQ ID NO:13. The primers and probes were prepared into 100 μM stock solutions for storage.
[0066] 2. Prepare other reagents: prepare PCR buffer, which contains 1.0mM MgCl2, dATP, dUTP, dGTP and dCTP each ...
Embodiment 2
[0072] Use the human MTHFR gene polymorphism detection kit prepared in Example 1 to detect the sample to be treated.
[0073] 1. Genomic DNA extraction from blood samples
[0074] Use the blood genome column extraction kit to extract the human blood genome according to the instructions. Use an ultraviolet spectrophotometer to detect the concentration of the obtained sample DNA solution, then dilute the sample DNA to 10ng / μl, take 2μl-5μl respectively and add them to the kit prepared in Example 1 and carry out the next step of PCR reaction.
[0075] 2. Take 2 μl of the DNA samples diluted in step 1 and add them to the reaction system of the kit of Example 1 of 23 μl in turn, and put them into a fluorescent quantitative PCR instrument, set the PCR reaction program as shown below and perform amplification reaction:
[0076] 50℃ for 2min, 95℃ for 10min;
[0077] 95℃ for 15s, 48℃~53℃ for 1min, 5 cycles;
[0078] 95°C for 15s, 60°C-62°C for 1min, 40 cycles; after each cycle, the...
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