摘要
目的:改进传统重叠延伸PCR方法,实现引入3个不同DNA突变位点的简便的多位点定点突变。方法:根据前期构建的包含人线粒体12S rRNA(NC 01290)3个热点突变位点的野生型质粒序列,利用Muta Primer 2.0软件设计针对3个热点突变位点的3对互补的定点突变引物,以野生型质粒为模板,结合重叠延伸PCR反应和冷冻析出法,产生同时包含3个突变位点的突变目的片段,酶切后克隆到载体中,测序确证是否突变成功。结果:DNA测序证实3个不同突变位点同时成功引入,定点突变载体构建成功。结论:用改进的重叠延伸PCR技术能简便、高效地获得多位点定点突变载体,在分子生物学领域有较高的使用价值。
Objective： To develop a simple multiple site-directed mutagenesis method by modifying the overlap extension PCR method to construct a vector containing three point mutations simultaneously. Methods： Three pairs of complementary overlapping mutagenesis primers were designed by using Muta Primer 2.0 software based on the sequence of the early wild-type plasmid containing three hot mutation points of human mitochondrion 12S rRNA （NC 01290）. The wild-type plasmid was used as a template, the DNA fragment including three point mutations was obtained through overlap extension PCR binding three pairs of complementary overlapping mutagenesis primers and frozen precipitation, then digested with restriction endonucleases and cloned into vector and sequenced to con firm whether the mutations were introduced. Results： DNA sequencing confirmed that the three different mutations were introduced. Mutant vector was successfully constructed. Conclusion： Modified overlap extension PCR can ob tain multiple site-directed mutagenesis carrier easily and efficiently and has a broad application prospects in the field of molecular biology.
出处
《生物技术通讯》
CAS
2014年第2期248-251,共4页
Letters in Biotechnology
基金
国家自然科学基金(81102516)
南华大学博士启动基金(2012XQD18)
关键词
重叠延伸PCR
多位点突变
定点突变
冷冻析出
overlap extension PCR
muhisite mutation
site-directed mutagenesis
frozen precipitation
