【摘要】 目的:建立重亚硫酸盐测序法(BSP法)定量分析ABO基因启动子区CpG岛的甲基化水平。方法:提取胃癌细胞株MKN45和KatoⅢ以及人全血基因组DNA,以重亚硫酸盐转化法修饰其DNA,应用Meth Primer软件在ABO基因启动子区非CpG位点区设计4对特异性甲基化扩增引物,将ABO启动子附近区域的CpG岛分成4个片段分别扩增,优化PCR扩增条件后进行T-A载体克隆和测序分析,采用BiQ Analyzer软件分析ABO基因启动子区CpG岛的甲基化水平。结果:PCR扩增获得了约2kb的完整CpG岛片段,共包含191个CpG位点,测序图谱证实亚硫酸盐转化修饰完全。MKN28细胞株整个CpG岛内191个检测的CpG位点全部甲基化,细胞株KatoⅢ甲基化比例为8.9%,随机标本DNA甲基化比例为30.4%。结论:建立的方法可检测出ABO基因启动子区CpG岛所有CpG位点的甲基化状况,定量分析其甲基化水平。更多还原

【Abstract】 Objective:To establish a stable sodium bisulfite sequencing(BSP) for analyzing ABO gene promoter CpG island methylation in quantitative.Methods: Genomic DNA were extracted from gastric cancer cell lines MKN45,KatoⅢ,and a normal individual.The DNA was converted unmethylated cytosines to uracils with sodium bisulfite.Four specific primers were designed in non-CpG sites region using Meth Primer software to amplify the whole CpG island in ABO promoter region with four amplification fragments.The PCR amplification conditions were optimal.Each fragment was ligated with TA cloning vector and sequenced.The level of CpG island methylation in ABO gene promoter region was analyzed by BiQ Analyzer software.Results: The length of 2kb CpG island were obtained by PCR amplification and spanned 191 CpG sites.The sequence profile showed that unmethylated cytosines completely converted to uracils by bisulfite-treated.All 191 CpG sites were methylated in the ABO promoter region of MKN28,while the methylated frequencies of KATOⅢ cell and normal individual were 8.9% and 30.4%.Conclusion: We have established a stable bisulfite-treated DNA sequencing method,which can detect the whole CpG island in ABO gene promoter and analyzed the methylation level of each CpG site in quantitative.更多还原