改进的PCR在抑癌基因启动子CpG岛甲基化检测中的应用

目的改进聚合酶链反应(PCR),并检验改进后的方法在甲基化检测中的可行性。方法培养人乳腺癌细胞株MCF-7,提取基因组DNA,利用亚硫酸氢盐测序法,用常规、降落及改进PCR分别扩增,比较扩增效果,PCR产物纯化回收后,TA克隆入载体pGEM-T,转化大肠杆菌(E.coliDH5α),筛选阳性克隆,经PCR扩增鉴定后获得阳性重组质粒,测序比对。结果改进后PCR与常规、降落PCR相比,扩增效率增高,二聚体及非特异性扩增减少;测序结果显示,MCF-7细胞基因组DNA中,RASSF lA基因启动子CpG岛是高甲基化的,E-cadherin基因启动子CpG岛未呈现高甲基化状态。结论改进后方法提高了以PCR为基础的甲基化分析的可行性,更适用于基因甲基化状态的检测,并为扩增富含CG的基因启动子区域提供参考。

Improvement and application of polymerase chain reaction in analysis of methylation of CpG island in promoter of anti-oncogene

Objective To develop a method for increasing the sensitivity and specificity of polymerase chain reaction(PCR) and discuss its feasibility in methylation analysis. Methods The genomic DNA was extracted from MCF-7 cells and treated by sodium bisulfite,the methylation status at RASSF1A and E-cadherin amplified with regular,touchdown and improved PCR respectively were detected by bisulfite sequenced,then the amplified DNA were subcloned into the TA cloning vector and sequenced. Results The improved method could provide the powerful feature that the CpG islands of RASSF1A but E-cadherin in MCF-7 cells was highly methylated. Conclusions Adding the primers at different time in PCR amplification and combining touchdown PCR can avoid nonspecific reactions and increase the specificity of PCR product,also the present method likely can be applied for amplification of other GC-rich genomic sequences.