用PCR法直接快速筛查重组阳性克隆  

A RAPID SCREEN FOR RECOMBINANT CLONES BY POLYMERASE CHAIN REACTION

作　　者：胡维[1] 向华[1] 周艳[1] 刘敬忠[1] 

机构地区：[1]首都医科大学附属北京红十字朝阳医院,北京100020

出　　处：《中国微生态学杂志》2000年第1期1-2,共2页Chinese Journal of Microecology

基　　金：北京自然科学基金

摘　　要：目的:应用PCR法快速筛查插入有苯丙氨酸脱氨酶(PAL)cDNA重组阳性克隆。方法:用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALcDNA3′端终止密码TAA处的引物。以灭菌吸头挑一单菌落加入PCR体系扩增。结果:在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实。结论:以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。Objective:To develop a polymerase chain reaction(PCR) technique for rapid screening of phenylalanine ammonia lyase(PAL) cDNA recombinant plasmid. Methods:PCR primers were designed so that one primer is complementary to the T 7 promoter of pET23b vector,and another primer complementary to the 3′ end of PAL cDNA.The recombinant colonies were transferred by using sterile pipet tip into the PCR reaction mixture directly.After PCR amplification,the PCR products were subjected to electrophoresis on 0 8% agrose gel. Results:2 positive clones with a correct direction by using the screening method were detected.The positive clones were further confirmed by DNA sequencing. Conclusions:Recombinant vectors in bacterial cells can be analyzed directly with the use of PCR. This convenient and timesaving procedure eliminates the need for preparation and purification of the plasmid.

关 键 词：基因克隆 苯丙氨酸脱氨酸 聚合酶链反应 

分 类 号：Q78[生物学—分子生物学]