平末端DNA随机连接构建重组质粒  

Construction of Recombinant Plasmids by Random Ligation of Blunt-Ended DNA

作　　者：查向东[1] 刘杨[1] 吴敌[1] 卢颖虎[1] 梁琳[1] 陈青峰[2] 

机构地区：[1]安徽大学生命科学学院,安徽合肥230039 [2]解放军电子工程学院门诊部,安徽合肥230032

出　　处：《生物技术通讯》2012年第1期109-111,共3页Letters in Biotechnology

基　　金：安徽省高校省级自然科学研究重点项目(KJ2010A025)

摘　　要：目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。Objective： To join different DNA fragments and clone the joint DNA.Methods： We randomly ligated the blunt-ended DNA fragments with T4 DNA ligase,cleaved the ligated molecules with the restriction endonucleases,then separated the hydrolysed products by agarose gel electrophoresis.We identified the specific band on the agarose gel,from which the DNA molecules were recovered,integrated into the linearized vector plasmid and transformed into the competent E.coli cells.Results： In this way we successfully joined the different DNA fragments and constructed the recombinant plasmids haboring the joint DNA.Conclusion： The stragety is simple,convenient and reliable,providing an alternative procedure for joining and cloning DNA which can be used in molecular biology research and gene engenering.

关 键 词：平末端DNA 随机连接 DNA拼接 基因克隆 

分 类 号：Q78[生物学—分子生物学]