Usage of homocysteine level prediction by polymorphic loci gene type, method and kit
A homocysteine, polymorphic site technology, applied in the field of medicine, can solve problems such as lack of predictability, and achieve the effects of alleviating patient pain, reducing medical costs, and preventing the increase
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Embodiment 1
[0053] Example 1: Determination of the C677T (Ala222Val, dsSNP ID: rs1801133) polymorphism site of the MTHFR gene and prediction of homocysteine levels
[0054] (1) Determination of the C677T polymorphism site genotype of the MTHFR gene
[0055] (1) Extract the genomic DNA of the host cell
[0056] (a) Add 30ml of erythrocyte lysate to the whole blood, shake slowly, and let stand at room temperature for 10 minutes. During this period, shake several times to completely lyse the erythrocytes;
[0057] (b) Centrifuge at 4°C and 2000 rpm for 10 minutes, remove the supernatant, break up the precipitated white blood cells on a rotary shaker, add 40 μl of protease and 50 μl of RNase, shake well, add 15 ml of white blood cell lysate, and mix Take it out after evenly bathing in 37°C water bath for 20 minutes, and put it in cold water;
[0058] (c) Add 4ml of cold protein precipitation solution, mix well and put it in the refrigerator at -20°C for 5 minutes, take it out and centrifu...
Embodiment 2
[0109] Example 2: A kit for determining the homocysteine level predicted by the genotype of the MTHFR C677T polymorphic locus
[0110] (1) The components of the kit: nucleic acid extraction reagents, PCR reaction reagents, MTHFR C677T polymorphic site genotype-specific primers, nucleic acid polymerase, restriction endonucleases, enzyme digestion reaction mixture and positive control template, Negative control templates were subpackaged and assembled into kits. The positive control templates include MTHFR C677T homozygous wild-type, heterozygous, and homozygous mutant positive control templates; the specific primers are primers capable of specifically amplifying at least the polymorphic site containing MTHFR C677T.
[0111] (2) Detection steps:
[0112] (1) Extract the genomic DNA of the host cell:
[0113] (a) Add 400 μl of erythrocyte lysate to a 1.5ml centrifuge tube, and add about 100 μl of fresh whole blood or anticoagulated whole blood. Water bath at 37°C for 5 minutes...
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