在单个PCR管内快捷完成定点突变

A Fast and Efficient Site-directed Mutagenesis Method in One Tube

采用大引物方法,利用质粒多克隆位点两侧的普通测序引物作为旁侧引物,在单个PCR管内,经2个步骤共34个循环进行定点突变.该方法通过优化模板和引物的量达到降低PCR循环次数,通过加入10个在68℃复性条件下的PCR循环达到增加突变效率而无需胶纯化.本方法达到平均62%的突变效率,而且全长扩增产物的产率很高.

Using normal sequencing primers adjacent to multiple clone sites of plasmid as flanking primers, a novel megaprimer method was performed in one tube with two steps and 34 cycles. The essential features of this protocol include an optimization of the template-primer amounts that allows the use of a reduced number of PCR cycles and the addition of 10 cycles at 68℃ annealing that can improve mutagenesis efficiency without gel purification. This method can reach 62 % of average mutagenesis efficiency and high yield of final products.