结合通用引物快速简便鉴定阳性克隆的PCR方法  

Rapid and Convenient Method for Positive Clone Screening and Identifying by PCR Using Universal Primers

作　　者：崔静[1] 黄爱龙[1] 张文露[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《生物技术通报》2008年第3期99-102,共4页Biotechnology Bulletin

基　　金：国家自然科学基金(30600520)

摘　　要：通用引物与载体多克隆位点两端序列互补,将目的片段构建入载体pGEM-T Easy中,利用载体通用引物M13F和M13R结合片段特异引物进行菌液PCR反应。用PCR产物电泳结果来筛选阳性克隆并鉴定目的片段插入方向,同时能有效对短插入片段重组子进行筛选与鉴定。最终以DNA测序结果来验证,与测序结果一致,显示通用引物PCR方法对阳性克隆的筛选和鉴定优于传统酶切和普通PCR鉴定方法,能够弥补传统方法的不足,且简便快速,可作为筛选和鉴定阳性克隆的有效手段。Constructing target gene into E.eoli vector pGEM-T Easy and amplifying bacterium liquid PCR reaction with universal primers,M13F and M13R,which are complemented with two terminal of vector multiple clone site. The electrophoresis result can be used to screen positive clone and identify the orientation of inserted fragment, especially to efficiently screen and identify short inserted fragment recombinants. Based on DNA sequencing method, compared and checked the result with enzyme digestion and common PCR. Comparing with the traditional enzyme digestion and common PCR,this new method can compensate the deficient of traditional methods. A rapid and simple PCR-based method for screening and identifying positive clone is described.

关 键 词：通用引物PCR 鉴定 插入方向 短插入片段 

分 类 号：Q78[生物学—分子生物学]