Kit for identifying polymorphic rs2274976 of human MTHFR gene by BsrI
A technology of rs2274976 and gene polymorphism, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of difficult popularization and use, expensive endonucleases, difficult selection and identification, etc., and achieve the effect of reducing costs
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Embodiment 1
[0085] 1 Materials and methods
[0086] 1.1 Main reagents and instruments
[0087] Reagents: 2×PCR mix (MBI Company), restriction endonuclease MspI (MBI Company), agarose (BBI Company), and primers synthesized by Shanghai Sangon Company.
[0088] Instruments: Model 9600 PCR instrument (PE Company), mini electrophoresis tank (Pharmacia Biotech, EPS1000), Gel Doc 2000 gel imager (Bio-RAD Company).
[0089] PCR product sequencing was completed by Shanghai Sangon Bioengineering Co., Ltd.
[0090] 1.2 Sequence search and primer design
[0091] Search the MTHFR gene sequence and SNP information on the NCBI website, determine the base variation information of the MTHFR polymorphic site in combination with relevant literature, and design primers, as follows:
[0092] The forward primer sequence is 5'-CTTTGCCCTGTGGATTGACC-3' (SEQ ID NO: 1),
[0093] The sequence of the reverse primer is 5'-GCAGTTGTCCAGTGGGAAGTCA-3' (SEQ ID NO: 2).
[0094] 1.3 Extract DNA from whole blood samples ...
Embodiment 2
[0112] Example 2 Determination of human MTHFR gene rs2274976 polymorphism in peripheral blood whole blood samples:
[0113] The steps in Example 1 are basically the same, except that the forward primer used in the PCR reaction is SEQ ID NO: 1, the reverse primer is SEQ ID NO: 7, the annealing temperature is 59 ° C, and the restriction endonuclease used It is HpaII (NEB Corporation).
[0114] result:
[0115] The obtained amplification product sequence is as follows (159bp, SEQ ID NO: 8):
[0116] ctttgccctg tggattgacc rgtggggaaa gctgtatgag gaggagtccc cgtcccgcac 60
[0117] catcatccag tacatccacg acaactactt cctggtcaac ctggtggaca atgacttccc 120
[0118] actggacaac tgcctctggc aggtggtgga agacacatt 159
[0119] When the MTHFR polymorphism contains a G allele, a 140bp product (SEQ ID NO: 9) can be formed after enzyme digestion, and the sequence is as follows:
[0120] crgtggggaa agctgtatga ggaggagtcc ccgtcccgca ccatcatcca gtacatccac 60
[0121] gacaactact tcctggtcaa cctggtggac ...
Embodiment 3
[0126] Example 3 Determination of human MTHFR gene rs2274976 polymorphism in peripheral blood clot specimen:
[0127] The steps are basically the same as in Example 1, except that the following method is used to extract DNA from the peripheral blood clot specimen as the DNA to be tested. In addition, the forward primer used in the PCR reaction is SEQ NO: 11 (tttgccctgtggattgacc), the reverse primer is SEQ NO: 12 (tctcgcattc tgggtggg), and the annealing temperature is 58°C. The restriction endonuclease was MspI (NEB Company).
[0128] DNA extraction:
[0129] Collect 400 μl of human peripheral blood in a common blood collection tube, and use the RelaxGene Rlood DNA System blood genome DNA extraction system from TIANGEN Company to extract the genomic DNA in the blood clot of the peripheral blood as the human genomic DNA template to be tested;
[0130] After the serum in the blood collection tube is separated out, separate the serum, and then follow the steps below:
[0131] G...
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