亚硫酸氢盐测序法检测上皮钙黏蛋白甲基化的优化探讨

Study on optimization of E-cadherin methylation detection by modified bisulfite sequencing

目的探讨改进的亚硫酸氢盐测序法在上皮钙黏蛋白甲基化检测中的可行性。方法提取雄性F344大鼠肾脏组织基因组DNA,亚硫酸氢盐化学转化,通过PCR扩增上皮钙黏蛋白基因启动子区特定的序列,以蓝白克隆斑筛选和测序。结果雄性F344大鼠肾脏上皮钙黏蛋白基因启动子区CpG岛的26个CpG位点均未发生甲基化。结论改进后亚硫酸氢盐测序法能明显减少非特异性扩增,提高PCR效率,更适于基因甲基化状态的检测。

Objective To investigate the feasibility of the modified bisulfite sequencing method in the E-cadherin methylation detection.Methods The genomic DNA was extracted from the kidney tissues of male F344 rats and chemically converted with bisulfite sodium.Then the specific sequence in the E-cadherin promoter region was amplified by PCR.The screening and sequencing were performed by the blue-white spot screening.Results No methylation change occurred at 26 CpG sites in the CpG islands of E-cadherin gene promoter in the kidney of male F344 rats.Conclusion The modified bisulfite sequencing method can obviously reduce the non-specific amplification,improves the efficiency of PCR and is more suitable for the detection of gene methylation status.