介绍一种PCR鉴定重组体DNA插入方向及转染的方法

介绍一种用ＰＣＲ方法鉴定重组体中插入片段的正、反连接方向及其是否导入细胞的方法．其原理是选择紧靠载体克隆位点上游的一段序列为上游引物，以扩增插入序列的上、下游引物分别作为下游引物进行ＰＣＲ．载体上游引物加插入序列的上游引物可扩出产物者为反向连接；加插入序列的下游引物可扩出产物者为正向连接．该法简单、快速，可广泛用于鉴定重组体中插入片段的正、反连接方向及基因转染时外源基因是否导入细胞内．

Identification of transfected gene and orientation of inserts in recombinants by PCR

A very simple and fast PCR method was developed to identify the orientation of inserts in the recombinants and whether the recombinants is transfected into the cells.The sequence near the polycloning site of vector was selected as upstream primer of PCR,and the upstream and downstream primer of the inserts as downstream primer separately.The forward recombinants can be amplified out PCR product using upstream primer of vector and downstream primer of the inserts,while reverse recombinants do using upstream primers of the vector and the inserts.The PCR identified orientation of the inserts was checked by conventional enzymatic digestion.This method was successfully used to identify reverse and forword pLXSN bcl 2 recombinants and the transfected pLXSN bcl 2 in PA317 cells.