用滚环扩增与大引物PCR法高效构建定点突变序列

A High Efficient Site-directed Mutagenesis Method by RCA and Megaprimer PCR

建立了一种简单、快捷的高效定点诱变方法。第一轮PCR反应中,上游引物和突变引物先以1∶10的比例进行不对称PCR,提高大引物突变比例。第二轮PCR反应以滚环扩增产生的单链DNA作为大引物PCR反应的模板,不需要优化PCR反应的条件,通过一般的反应条件就可得到大量PCR产物,并且诱变的成功率可达100%。

In this research, a simple, convenient and high efficient method for site -directed mutagenesis was established. In the first PCR reaction, an asymmetrical PCR was conducted by using the forward/mutagenic prime pair at the ratio of 1：10 in order to enhance the proportion of mutagenie megaprimer. In the second PCR reaction, the PCR template was the single - stranded DNA produced by rolling circle amplification （RCA）. Thus, large amount of PCR products could be obtained by routine approaches without optimization of PCR conditions. The success rate of mutagenesis could reach 100%.