一种载体构建的新方法:重组融合PCR法

A Novel Method for Vector Making: Recombination-Fusion PCR

本研究介绍一种多目的片段一步重组克隆的载体构建的新方法。该方法要求在PCR引物设计时,在第一个目的片段上游引物的5'端和最后一个目的片段下游引物的5'端各设置16bp与线性化目标载体末端同源的序列,再在各目的片段之间的引物上各设计25bp的重叠序列,以便进行融合PCR将多片段扩增成融合片段。此方法将融合片段与线性化骨架载体进行一步重组、转化和重组子鉴定。该方法只要求骨架载体上有一个特异性酶切位点,特别适用于插入片段中含有较多限制性酶切位点的载体构建;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;该构建方法还可以实现多基因的无缝融合构建多顺反子,在构建原核基因表达载体、叶绿体表达载体、线粒体表达载体方面有着明显优势。本研究以水稻胚乳特异性表达载体PGt1-mGFP-pSB130为例,介绍此载体构建方法。

This study introduced a novel method for constructing vectors by combination of recombination and fusion PCR. The core of this method is the special rules for primer design： 16 bp homologous sequences, around the restriction enzyme sites used to linearizing the target vector, was designed before the 5＇ end of the primer F of the first target fragment and the primer R of the last target fragment. 25 bp over lapped sequences of closing target fragment was designed before the 5＇ end of other primers. With this special design of primers, we can construct a vector with two or more target fragments in one step cloning. With this method only one specific restriction site was needed in the target vector, it＇s easy to insert DNA fragments with many restriction sites to the target vector. Moreover, this method can also introduce site-directed mutagenesis into target DNA. In addition, this method can fusion several genes without any unwanted sequences between two target fragments. It is appro-priate for prokaryotic gene expression construct, chloroplast expression construct and mitochondrial expression con-struct. Rice endosperm-specific expression vector, PGt1-mGFP-pSB130 was constructed in this article to show the detail of this novel method.