【摘要】目的 比较普通聚合酶链反应(PCR)与降落聚合酶链反应(Touchdown PCR，TDPCR)在临床医学科研中的价值。方法 以人类外周血基因组DNA为模板，设计VHL基因3个外显子的3对引物，根据普通PCR及TDPCR原理设计包括3个外显子片段在内的PCR程序，通过试验选择PCR最佳反应条件，在同一程序中分别对3个片段进行扩增，琼脂糖凝胶电泳检测扩增产物，纯化后PCR产物测序分析两种试验方法的差别。结果 电泳检测及纯化后DNA产物测序分析均显示TDPCR扩增产物条带特异性、效率较普通PCR扩增产物高。结论 成功建立了TDPCR方法，TDPCR方法较普通PCR更为高效实用，为临床基因突变筛查研究提供了快速可靠的手段。
【关键词】 基因 聚合酶链反应 降落聚合酶链反应
Application of Ordinary PCR and TDPCR to Clinical Medicine
Wang Nan, Wang Yangmin, Zhang Bin
(Clinical Medical College of Lanzhou University, Lanzhou, 730000, China; Department of Urology, Lanzhou General Hospital of Lanzhou Military Region, PLA, Lanzhou 730050, China)
Abstract： Objective To compare the ordinary polymerase chain reaction (PCR) and touchdown (TD)PCR in clinical medical research. Methods We used genomic DNA from human peripheral blood as templates and designed three pairs of primers of VHL gene three exons. On the basis of the principle of PCR and TDPCR, we designed the programs, including the three exons. We chose the better reaction conditions through the PCR tests. The same programs were carried out on three fragments. The PCR products were detected by gel agarose electrophoresis. We analyzed the difference between the two methods by sequencing purified PCR products. Results It was indicated that TDPCR was more specific and effective than ordinary PCR, according to electrophoresis detection and sequencing analysis. Conclusion TDPCR, which acts as a rapid and reliable method, is more efficient than ordinary PCR for clinical gene mutation screening.
Key words： gene; polymerase chain reaction; touchdown polymerase chain reaction
自从20世纪80年代中期以来，聚合酶链反应(Polymerase chain reaction，PCR)作为一种体外扩增DNA的方法，在分子生物学、法医学及一些人类基因疾病诊断等方面起到越来越重要的作用。PCR所具有的3个突出的特点(即选择性，特异性和快速性)，使得DNA的克隆和操作变得十分简单。目前在一些临床基因突变筛查研究中，PCR作为基本实验手段，得到了广泛的应用,尽管如此，许多PCR实验中不但经常出现假阳性，而且实验结果往往并不太令人满意，也会造成无PCR产物，或电泳中出现大量非特异性二聚体条带。降落聚合酶链反应(Touchdown PCR，TDPCR)，最初是用于克服PCR中的假引发，作为一种
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