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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 and verify the methylation of CMV promoter. Methods The HepG2 DNA of pEGFP-C3 recombinant human hepatoma cell line and the chemical modification of sodium bisulfite were extracted. Specific primers were designed according to the CMV promoter sequence of the modified plasmid gene. The PCR products were cloned and sequenced. Based on quantitative. Results The methylation level of about 600bp in CMV promoter region of pEGFP-C3 plasmid gene could be accurately quantified. The results were consistent with those of the standard and the results of repeated measurements were stable. Conclusion The improved sodium bisulfite sequencing method can significantly reduce non-specific amplification, improve PCR efficiency, and is more suitable for the detection of gene methylation status.