一种快速简便的缺失突变方法

A rapid and simple method of mutagenic deletion

为了构建能表达稳定的、不易降解的ＴＭ－ＴＮＦ突变体（ＴＭ－ＴＮＦｍ）的基因，本文用改良的Ｓ－ＰＣＲ和ＯＥ－ＰＣＲ两种定位突变技术，成功地构建了缺失３６个碱基的ｐＢＳＫ－ＴＭ－ＴＮＦ突变重组体。与ＯＥ－ＰＣＲ技术（用两对引物进行两次ＰＣＲ反应）相比较，Ｓ－ＰＣＲ技术（用一对引物做一次ＰＣＲ反应）具有快速、经济、简便等优点，但其突变率较ＯＥ－ＰＣＲ低。

In order to obtain a gene capable of expressing stable, nonsecretable transmembrane TNF mutant(TM TNFm), we adopted two different techniques for site directed mutagenesis, in the present study, namely, modified single step PCR(S PCR) and overlap extension PCR (OE PCR). With both of these methods we succeeded in constructing the pBSK TM TNF mutant recombiant as a result of deletion of 36 bp nucleotides from the gene of tumor necrosis factor α(TNF α). It was shown that the technique of S PCR in which only one single pair of specific primers and one single step of PCR are needed, seemed to be more rapid, more convenient and less expensive than that of OE PCR in which two pairs of primers and two times of PCR are necessary, although S PCR was shown to have a lower mutagenic rate than OE PCR.