建立基于新型基因转换系统的检测TP53基因（R175H）早期突变的新方法

发布时间：2018-03-26 13:16

本文选题：高保真DNA聚合酶　切入点：限制性内切酶　出处：《苏州大学》2016年硕士论文

【摘要】：目的建立一种新型基因转换系统,用于检测TP53基因(R175H)早期突变,为肿瘤的早期筛查提供依据。方法构建TP53基因野生型DNA模板(含NarI酶切位点)和突变DNA模板。将TP53基因野生型基因片段与突变型基因片段按照野生:突变(W:T)=1:0,100:1,300:1,1000:1的比例混合,使用高保真DNA聚合酶进行PCR扩增,过程中不断添加限制性内切酶进行酶切消化,并对其产物进行PCR扩增后再行测序。结果琼脂糖凝胶电泳结果显示,纯野生型基因片段的PCR产物可以被NarI限制性内切酶酶切,而其余三种混合比例中因含有突变型基因片段,其PCR产物有不能被酶切的片段产生,使得突变型基因获得了大量扩增,经测序后亦证实上述结果。结论在高保真DNA聚合酶及低温限制性内切酶的基础上,联合特异性引物构成的基因转换系统检测早期TP53基因(R175H)突变是可行的,这个基因转换系统可能在基因早期筛查等方面具有较大的实际应用价值。
[Abstract]:Objective to establish a novel gene transformation system for detecting the early mutation of TP53 gene r175H. Methods the wild type DNA template of TP53 gene (including NarI restriction site) and the mutant DNA template were constructed. The wild type gene fragment of TP53 gene and the mutant gene fragment were mixed according to the ratio of 1: 100: 100: 100: 1: 300: 11 000: 1 of TP53 gene. High fidelity DNA polymerase was used for PCR amplification. Restriction endonuclease was added in the process of digestion, and the products were amplified by PCR and sequenced. Results agarose gel electrophoresis showed that, The PCR products of pure wild-type gene fragments can be digested by NarI restriction endonuclease, but the other three kinds of mixed proportion contain mutant gene fragments, so the PCR products can not be digested, which makes the mutant genes obtain a lot of amplification. Conclusion on the basis of high fidelity DNA polymerase and low temperature restriction endonuclease, it is feasible to detect the early TP53 gene r175H mutation by a gene conversion system composed of specific primers. This gene conversion system may be of great practical value in early gene screening.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.43

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