重组PCR技术的应用及优化

Applications and Optimization of Recombinant PCR

重组PCR是通过DNA重叠序列的衔接作用,使多个DNA分子融合在一起的体外扩增技术。它使基因全序列的拼接、基因融合、基因破坏及启动子交换等DNA操作变得简单易行。如今重组PCR已成为DNA分析的有效利器。本研究通过重组PCR在分子进化、基因敲除及基因敲入、启动子研究和转基因植物转化载体的构建等方面的实际应用,分析了该技术的影响因素,并针对引物设计、DNA碱基重叠长度、温度参数等重要反应条件提出了优化方案。

The recombinant PCR is an efficient method in vitro to fuse several DNA molecules by linker sequences. It facilitates a wide range of DNA manipulation techniques, including the joint of the full length of gene sequence, gene fusion, gene disruption and promoter exchange. Up to date, recombinant PCR has become an effective tool for DNA analysis. In this study, the influential factors of recombinant PCR were analyzed and discussed based on its applications in molecular evolution, gene knock-out and knock-in, and the construction of vectors for transgenic plants. The optimized designs for the important parameters of recombinant PCR were proposed, including the primer design, the length of overlapping sequence and the temperature parameters in reaction.