Establishment of methylation specific pyrosequencing for RARβ2 gene promoter methylation detection
AIM: To establish a methylation specific pyrose-quencing method for detecting RARβ2 methylation in the gene promoter region. METHODS: Genomic DNA was extracted from human normal whole blood. Targeted gene fragment was amplified by composite nested PCR and cloned into pMD 18-T vector for constructing control plasmid. Five methylation loci were detected in RARβ2 gene promoter CpG island by pyrosequencing after bisulfite treatment, and the results were confirmed by dideoxy chain terminant sequencing method. The degree of methylation was detected in different proportion admixture of control PCR products and then the calibration curve was made. RESULTS: Negative control and positive control plasmids for methylation specific pyrosequencing were successfully constructed. The degree of methylation was 0% in negative control and 100% in positive control. By detecting mixed samples, we found a methylation-linear relationship between the frequency of complex, with the linear correlation greater than 0.99, the slope about 1, and the standard deviation in all sites about 1%. CONCLUSION: The methylation specific pyrosequencing method for detecting gene promoter methylation is successfully established.
NETL
NSTL
万方数据
维普
