降落PCR及克隆测序法检测DNA甲基化的实验研究

目的 探索优化PCR及测序法检测DNA甲基化的实验条件,检测肺癌hsa-mir-34b(mir-34b)基因启动子DNA甲基化谱。方法 肺癌细胞株A549 DNA进行亚硫酸盐处理后,设计引物扩增mir-34b基因启动子序列,使用普通PCR、巢式PCR和降落(Touchdown)PCR方法以及不同DNA聚合酶进行扩增,甲基化谱检测采用PCR直接测序和克隆后测序两种方法。结果 降落 PCR与巢式PCR方法相结合优于普通PCR及单独的巢式PCR或降落 PCR。HotStart酶优于普通Taq酶及Ex Taq酶。直接PCR测序法的测序图普遍出现套峰难以判读,PCR产物克隆后测序可见清晰的测序峰,甲基化位点可明确判读,并可以结合测序克隆数目提高甲基化检测敏感度。结论 对于重亚硫酸盐处理的DNA甲基化状态检测,推荐使用HotStart酶、降落 PCR与巢式PCR相结合、克隆测序法检测基因甲基化谱,序列图明确且敏感度高。

The detection of DNA methylation using touchdown PCR and cloning-based sequencing methods

Objectives Explore the best experiment condition for touchdown PCR and clone - based sequencing to detect the methylation status of hsa - mir - 34b gene promoter. Methods After the bisulfite treatment of the DNA extracted from lung cancer cell line A549, the primer was designed to amplify the promoter region of mir- 34b gene. General PCR,nest PCR and touchdown PCR were employed to amplify the target fragment with different DNA polymerase. Then direct sequencing and cloning - based sequencing methods were performed to detect the methylation status. Results Compared to general PCR, touchdown PCR and nest PCR was used alone, touchdown PCR together with nest PCR was much easier to amplification ; HotStart Taq was the suitable DNA polymerase in the PCR reaction system. Compared to the direct sequencing, cloning - based sequencing could find the methylation status more clear. Conclusions HotStart Taq DNA polymerase, touchdown PCR together with nest PCR and cloning - based sequencing methods were recommend to be used to detect the methylation status after the DNA underwent the bisulfite treatment, by which we could obtain the methylation patterns clearly with high sensitivity.