DNA克隆载体的“三片段克隆法”改造策略

A General Method for Direct DNA Vector Modification

将一个或多个DNA片段克隆至载体是分子生物学研究中最常用的技术之一.本研究使用新颖的Red/ETDNA重组克隆技术原理一步法将结合转移片段oriT,阿普霉素抗性基因Am和链霉素抗性基因(rpsL)克隆至常用的克隆载体pBluescriptKS(-),同时去除载体的氨苄青霉素抗性基因(bla).此"三片段克隆法"简便,快速,避免了经典的克隆策略常常难以寻找合适的限制性内切酶,长片段PCR、暴露于紫外线和凝胶回收等步骤所可能引入的碱基突变等等难以克服的问题,有可能成为通用的克隆策略.所得到的结合转移载体在通过结合转移将外源片段从大肠杆菌等转移至放线菌方面将有着广泛的用途.

Cloning one or more DNA fragments into a vector is the one of most commonly used techniques in molecular biology. Classic cloning strategy, when dealt with complicated cloning steps, like multiple fragments insertion, selective marker exchange, are often troubled by the choice of restriction enzymes, the base mutations caused by long fragment PCR, UV exposure and gel purification. We made use of the Red/ET technology to clone two fragments into the vector while remove the unnecessary vector part in a single step. This three pieces cloning manipulation is straightforward and convenient, with the potential to be a general cloning strategy for vector engineering.