A method for quantitative detection of methylation by pyrosequencing
A quantitative detection and pyrosequencing technology, applied in the biological field, can solve problems such as increased analysis costs, complex types of DNA templates, and difficulty in designing assays, and achieve the effect of increasing the length of the assay and broadening the scope of the analysis
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[0031] The method for analyzing the five methylation sites of the CpG island of the RARβ2 gene promoter in human samples, the specific methods include:
[0032] (1) Genomic DNA in peripheral blood was extracted from the blood sample using traditional protein kinase K and phenol / chloroform extraction methods;
[0033] (2) Genomic DNA modified by sodium bisulfite: Genomic DNA was treated with sodium bisulfite according to the operating procedure provided by CpGenome Turbo Bisulfite Modification Kit (Millipore Company), and the modified DNA was purified and recovered;
[0034] (3) PCR amplification: PCR primers: 5'-biotin-GTTGTTTGAGGATTGGGATG-3', 5'-ATACCCAAAACAAACCCTACTC-3' and 200mg of genomic DNA treated with sodium bisulfite, 0.2mM dNTP, 1UTaq DNA polymerase, 1 × Amplification buffer, 1.8mM MgCl 2 The 50 μL PCR amplification system was subjected to 0 amplification, and the amplification conditions were: initial denaturation at 95°C for 5 minutes; 40 thermal cycles: denaturat...
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