一种用高拷贝质粒载体制备BAC载体基本功能基因的新方法

[目的]简便有效地解决BAC载体序列因单一拷贝数带来的制备以及下游亚克隆操作的困难。[方法]选择适当的单一酶切位点,将单拷贝BAC载体质粒序列全部插入高拷贝质粒pUC119载体的适当位点。[结果]BAC载体基本功能基因序列在新质粒pUC119-BAC中失去了单一拷贝数控制功能,以高拷贝形式进行复制,利用单一酶切,可将BAC载体基本功能基因序列完整切下来,自连后重新恢复严格的单拷贝控制功能。[结论]利用高拷贝pUC119-BAC质粒,实现了BAC载体的基本功能基因序列高拷贝的复制和扩增,可以方便地用于分子克隆化重组病毒转移载体的构建或BAC文库的构建。

A New Method for the Preparation of BAC Vector DNA With High-copy Plasmid

[Objective]The aim of this study was to provide a method for solving problems in preparing BAC vector with high-copy plasmid pUC119-Bluelox BAC.[Method]With selecting a proper single restriction site,sequences of a single copy BAC vector plasmid were inserted into proper site of high-copy plasmid pUC119 vector.[Result]The gene sequence of BAC vector basic function lost control function of single copy number in new plasmid pUC119-BAC but copied through high-copy form.The gene sequence of BAC vector basic function was complete cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection.[Conclusion]The high-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector,besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.