改进的亚硫酸氢钠测序法检测HepG2 RASSF1A基因CpG岛甲基化状态

目的 改进亚硫酸氢钠测序法，并检验改进后方法在甲基化检测中的可行性。方法 提取人肝癌细胞株HepG2基因组DNA，亚硫酸氢钠化学修饰，针对修饰后Ras相关家族1A基因（RASSF1A）启动子序列设计特异引物并结合沉降PCR扩增，T-A载体克隆、测序。结果 HepG2细胞RASSF1A基因启动子CpG岛的16个CpG位点均被甲基化。结论 改进后亚硫酸氢钠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。

Analysis of Methylation at CpG Island of RASSF1A in HepG2 by Modified Bisuifite Sequencing

Objective To test the feasibility of modified bisulfite sequencing in methylation analysis.Methods The genomic DNA was extracted from the HepG2 cells and treated by bisulfite sodium.The methylation at Ras association domain family 1A gene(RASSF1A) was detected by means of bisulfite sequencing and "touchdown" polymerase chain reaction,then the amplified DNA was subcloned into the TA coloning vector and sequencing.Results The CpG islands of RASSF1A in HepG2 cells were completely methylated.Conclusion The modified bisulfite sequencing can avoid nonspecific reactions and increase the specificity of PCR product,also the present method likely can be applied to other GC-rich genomic sequences.