【摘要】 目的:通过使用优化后的定点突变三种方法分别对一个新基因重组载体进行定点突变研究,比较这几种定点突变方法的优缺点。方法:重叠延伸 PCR 法使用 Stratagene 在线定点突变引物设计程序从而使引物设计简化而可靠;M utaBEST 定点突变试剂盒采用胶纯化试剂盒代替说明书推荐的酚-氯仿抽提质粒的方法以提高回收率;使用 Prim eSTA R 高保真 D N A 聚合酶和超级感受态试剂盒代替 Q uikchange 定点突变试剂盒的相应组分可使产物不受影响同时降低试验费用。结果:三种方法都能够获得单碱基突变重组载体。结论:Q uikChange 突变策略通过改进是一种高效、简洁、经济和可靠的定点突变方法。更多还原

【Abstract】 Objective: Analysis of improved three site-directed mutagenesis methods in a novel gene recombinants construction. Methods: Overlapping extension PCR, MutanBest kit and Quikchange site-directed mutagenesis kit were used to construct mutants con-taining a single different residue from wild type gene. By Stratagagene primers design on-line, PCR was simplified. Comparing with PrimeSTAR polymerase and Ultra-Super competent cell kit substitution, Quikchange kit protocol was more economical. Results: Objec-tive recombinants were successfully gained by all three methods, so recombinant vectors could be utilized in further test. Conclusions: With two key elements substitution, Quikchang site-directed mutagenesis protocol could be improved to an effective, convenient, simple and economic approach.更多还原