一种不依赖酶切位点的分子克隆方法

A Novel Restriction Enzyme Sites-independent Method of Molecular Cloning

本研究介绍一种分子克隆的新方法。该方法要求在PCR引物设计时,目的片段上游引物的5'端和下游引物的5'端各设计约25 bp与目标载体末端同源的序列,或目的载体反向游引物的5'端和正向引物的5'端各设计约25 bp与目标片段同源的序列,以便进行融合PCR,PCR反应扩增目的片段后,与载体进行融合PCR。用DpnⅠ消化原始甲基化模板,然后进行转化和重组子鉴定。结果表明利用该方法成功将目的片段插入载体。证明这是一种简便、通用、高效并值得推广的分子克隆的新方法。该方法不需要骨架载体上的特异性酶切位点,特别适用于插入片段中无限制性酶切位点的载体改造;该方法还可以引入定点突变,可便捷构建分析启动子功能等需引入定点突变的载体;该分子克隆方法还可以实现基因的无缝克隆。

This study introduced a novel method of molecular cloning. The key of this method is the special rules for primer design： About 25 bp sequences homologous with the target vector were designed before the 5＇ end of the primer F and the primer R of the target fragment, or about 25 bp sequences homologous with the target fragment were designed before the 5＇ end of the primer F and the primer R of the target vector. With this special design of primers, fusion PCR with target vectors were performed after the target fragments were amplified by PCR, the next step is to digest methylated template by Dpn I and transform the PCR products and finally identifying the transformants. With this method DNA fragments were successfully inserted into vectors. Scores of experiments have proved that it is a simple universal high-throughput method that is worth popularizing to molecular cloning. With this method specific restriction site was not needed in the target vector, and it＇s easy to insert DNA fragments with any sites to the target vector. Moreover, this method can also introduce site-directed mutagenesis into target DNA especially in the analysis of promoter function. In addition, this method can insert DNA fragments into vector without any unwanted sequences between two target fragments.