焦磷酸测序检测RARβ2基因启动子甲基化方法的建立

Establishment of methylation specific pyrosequencing for RARβ2 gene promoter methylation detection

目的:建立基因启动子甲基化的焦磷酸测序检测方法,为基因启动子甲基化标志物的检测奠定基础.方法:从正常人全血中提取基因组DNA.采用复合巢式PCR的方法扩增目的基因,克隆构建对照标准品质粒.使用甲基化特异性焦磷酸测序检测RARβ2基因启动子CpG岛的5个甲基化位点,双脱氧测序验证.将阳性和阴性对照标准品质粒PCR扩增产物按不同比例混合后进行焦磷酸测序,检测每个点的甲基化程度,制作校正曲线.结果:构建了甲基化特异性焦磷酸测序阴性对照和阳性对照标准品.经焦磷酸测序检测后,阴性对照标准品甲基化程度为0%,阳性对照标准品甲基化程度为100%,与双脱氧测序结果一致.经对混合样品检测,甲基化频率符合线性关系,线性相关性大于0.99,斜率约为1,并且所有位点的标准偏差约为1%.结论:成功构建了甲基化特异性焦磷酸测序检测RARβ2启动子区域甲基化阳性对照和阴性对照标准品质粒.建立了甲基化特异性焦磷酸测序检测基因启动子甲基化的方法.

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.