DNA甲基化PCR方法的优化

【目的】优化DNA甲基化PCR的反应体系及扩增参数，为基因甲基化的研究建立一个最佳的PCR方法。【方法】以MHCC-97H肝癌细胞株AFP基因启动子区为模板，分析DNA甲基化反应体系中Mg^2＋、TaqDNA聚合酶用量、加入时间、退火温度及循环方式对甲基化PCR扩增的影响。【结果】反应体系20μl：模板DNA（MoDNA），2μl；正向引物，1μl；反向引物，1μl；10×PCR Buffer，2．5μl；2mmol／LdNTPs，2．5μl；Taq酶，0．4μl；MgCl2，2μl；H2O，8．6μl。PCR程序采用Touch Down PCR。【结论】优化了适用于AFP基因甲基化PCR的反应体系，为利用甲基化PCR分析方法研究肝癌等肿瘤发生机制奠定基础。

Optimization of PCR conditions in DNA methylation assay

[ Objective] To establish a optimal procedure for DNA methlation PCR analysis, the optimized reaction conditions and methods of PCR in the assay has been studied. [Methods] The promoter region in AFP gene of MHCC-97H HCC cell lines were used as the template. The affecting factors in PCR include Mg^2＋ , TaqDNA polyrnerase, annealing temperature and procedure et al. [Results] In the 20 μl amplification reactions system containing template DNA 2 μl, 10 × PCR buffer （Mg^2＋ free） 2.5μl, MgCl2 2μl, dNTPs 2.5 μl, Taq DNA polymerase 0.4μl, sense primer 1 μl and antisense primer 1μl. The optimal amplified procedure was studied and the Touch Down PCR had been adopted. [ Conclusions] A reaction system and amplified procedure suitable for methylation assay were established. It is the foundation for the study of mechanism of tumorigenesis.