pEGFP基因启动子区甲基化的梯度降落PCR定量检测法

Quantification of Methylation at CMV Promoter of pEGFP-C3 Vector by Gradient Touch-Down PCR Sequencing

目的改进亚硫酸氢钠测序法,并在CMV基因启动子甲基化检测中进行验证。方法提取pEGFP-C3质粒重组人肝癌细胞株HepG2 DNA,亚硫酸氢钠化学修饰,针对修饰后质粒基因CMV启动子序列设计特异引物并结合梯度降落PCR扩增,T-A载体克隆、测序,目标区域甲基化定量。结果 pEGFP-C3质粒基因约600bp的CMV启动子区甲基化水平可以精确定量,检测结果与标准品一致,重复测量结果稳定。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。

Objective To improve the sodium bisulfite sequencing method and validate it in quantification of methylation in CMV promoter. Methods DNA was extracted from recombinant HepG2 hepatoma cell line with plasmid pEGFP-C3, and chemically modified with sodium bisulfite, the gene-specific primers.were designed according to the modified CMV promoter sequence and conducted PCR amplification with gradient touch-down PCR, then the DNA methylation level in the target areas was quantitated after T-A cloning and sequencing. Results The DNA methylation level of the 600 bp CMV promoter within pEGFP- C3 plasmid could be quantified accurately, and be consistent with that of the methylated DNA standards, repeated measurements indicated stable quantification result with this method. Conclusion The improved sodium bisulfite sequencing method can reduce non-specific amplification significantly and improve PCR efficiency, therefore has more potential for quantitative detection of gene methylation status.