利用重叠延伸PCR技术进行定点突变研究

[目的]建立一种高效、便捷、经济的DNA定点突变方法。[方法]以重组人组织型纤溶酶原激活剂rPA(Reteplase)基因为模板,采用重叠延伸PCR技术对3个位点进行定点突变,将突变基因片段克隆到克隆载体pEASY-Blunt上,并通过测序验证突变结果。[结果]测序结果表明3个位点的突变结果与预期完全一致,即第10位引入单个碱基A、第137位碱基C突变为G以及第686位碱基G突变为A,通过重叠延伸PCR技术一次引入3个突变碱基,100%的实现目的位点的定点突变。[结论]该研究成功实现目的位点的定点突变,为rPA基因的进一步克隆和功能研究奠定了基础。同时也表明重叠延伸PCR技术是一种高效、便捷、经济的DNA定点突变方法。

Site-directed Mutagenesis Based on Overlap Extension PCR

[Objective] To establish an efficient,convenient and economical method for site-directed mutagenesis.[Method] The target mutation was introduced into primers designed by DNAMAN5.0 software.Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator(Reteplase).The mutation gene cloned it into pEASY-blunt simple cloning vector for sequencing.[Result] The sequencing results showed that three site mutations were fully consistent with the expected results(10th site had been added a base-pair of A,C had been changed into G at 137th site,G had been changed into A at 686th site).Three site mutations were introduced by using overlap extension PCR on one-step.The overall rate of obtaining the mutant sites was 100%.Site-directed mutagenesis will clone the recombinant Human Tissue type plasminogen activator and laid the basis for the functional study.[Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient,convenient and economical DNA-directed mutagenesis method.