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

作　　者：江艳[1,2] 王先良[2] 王春晖[3] 王小利[4] 杨怡姝[4] 段小丽[2] 聂静[2] 王菲菲[2] 张金良[2] 

机构地区：[1]厦门大学海洋与环境学院,福建厦门361005 [2]中国环境科学研究院环境污染与健康研究室,北京100012 [3]安徽医科大学公共卫生学院,安徽合肥230032 [4]北京工业大学生命科学学院,北京100022

出　　处：《环境与健康杂志》2010年第5期396-399,共4页Journal of Environment and Health

基　　金：国家自然科学基金资助项目(20907047);中央级公益性科研院所基本科研业务专项(2008KYYW05)

摘　　要：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.

关 键 词：基因 亚硫酸氢钠测序法 甲基化 梯度降落PCR CMV启动子 pEGFP—C3 

分 类 号：R994.6[医药卫生—毒理学]