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DNA甲基化PCR方法的优化
引用本文:马勇,俞媛,王玮,陈立军. DNA甲基化PCR方法的优化[J]. 武警医学院学报, 2008, 17(12): 1045-1047
作者姓名:马勇  俞媛  王玮  陈立军
作者单位:1. 武警医学院科研部,天津,300162
2. 天津市天和医院检验科,天津,300050
3. 武警医学院生化教研室,天津,300162
摘    要:【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。

关 键 词:AFP基因  甲基化PCR  条件  优化

Optimization of PCR conditions in DNA methylation assay
MA Yong,YU Yuan,WANG Wei,CHEN Li-jun. Optimization of PCR conditions in DNA methylation assay[J]. Acta Academiae Medicinae CPAPF, 2008, 17(12): 1045-1047
Authors:MA Yong  YU Yuan  WANG Wei  CHEN Li-jun
Affiliation:MA Yong, YU Yuan, WANG Wei, CHEN Li-jun (Scientific Research Department, Medical College of Chinese People's Armed Police Force, Tianjin 300162, China)
Abstract:[ 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.
Keywords:AFP gene  Methylation-specific PCR  Conditions  Optimization
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