一种改进的从细菌人工染色体中亚克隆DNA片段的方法  

An Improved Method for Subcloning DNA from BACs

作　　者：蔡东晖[1] 石建军[2] 叶荣[1] 陈学进[3] 

机构地区：[1]苏州大学生命科学学院,江苏苏州215123 [2]中国科学院上海生命科学研究院,上海200031 [3]上海交通大学医学院附属新华医院发育生物学研究中心,上海200092

出　　处：《苏州大学学报（医学版）》2008年第2期218-221,225,共5页Suzhou University Journal of Medical Science

基　　金：国家科委973资助项目(001CB509903;001CB509904)

摘　　要：目的运用基于PCR的重叠区延伸法构建缺口修复载体,通过同源重组的方式从细菌人工染色体(BACs)中亚克隆DNA片段。方法用基于PCR的重叠区延伸法将线性化质粒骨架分子、较长的上游和下游同源臂(200～500bp)串联起来构建成缺口修复载体。将此载体电击转入含有BAC的EL350菌中,经热激诱导Red重组酶瞬时表达,使缺口修复载体与BAC发生同源重组。结果重叠区延伸法构建的两个缺口修复载体分别成功地从BAC中亚克隆得到目的DNA片断。结论该方法省去了酶切、连接、转化等繁琐的克隆步骤,大大缩短了实验周期,提高了实验效率,为基因组BAC测序过程中的缺口封闭及复杂的打靶载体构建提供了较为便捷的方法。Objective To subclone DNA from BACs （bacterial artificial chromosomes, BACs） by homologous recombination between the BACs and gap repairing vector which was built by way of PCR-based fusion. Methods Two longer homology arms （200-500 bp） were added to the two ends of a plasmid backbone by PCR-based fusion. Large amounts of linear gap repairing vectors were obtained by the following PCR amplification then electroporated into EL350 cells. DNA fragme with primers. The gap repairing vectors were nts were successfully subcloned from BACs by homologous recombination between the linear gap repairing vector and BACs after heat shock in- duction of Red recombination proteins. Results Both gap repairing vectors, constructed during PCR-based fusion,successfully subcloned the desired DNA fragments from BACs. Conclusion This method that doesn＇t require any restriction endonuclease operation is fast, efficient and makes it possible to subclone DNA fragments in a short period. This method should facilitate the subcloning DNA from BACs for the purposes like gap closure in BAC-based genomic sequencing and complicated targeting vector constructs.

关 键 词：细菌人工染色体 缺口修复载体 PCR融和 重组 

分 类 号：R394.8[医药卫生—医学遗传学]