PCR重叠延伸法结合分段克隆技术构建高GC含量基因突变载体

Mutate gene of high GC content with gene splicing by overlap extension and fragmentation cloning method

目的:构建人类KAP-1基因定点突变载体,为研究该基因的功能奠定基础。方法:综合运用PCR重叠延伸法和分段定向克隆技术。结果:DNA测序结果表明定点突变载体构建成功。结论:成功构建高GC含量(局部高达80.50%),全长为2 500bp的人类KAP-1基因定点突变载体pCMV6-En-try-KAP-1,为全面深入研究KAP-1基因的功能奠定了基础。

Objective： To construct the mutation vector of human KAP-1 gene, and to provide sci- entific base for further study on the function of KAP-1. Methods=Overlap extension PCR （SOE PCR） and fragmentation cloning method were applied in this study. Results： DNA sequence demonstrated that the mutant vector had been recombined. Conclusions： The recombinant eukaryotie plasmid pCMV6-Entry-KAP-1 with high GC content （80.5% in the highest fragment） is successfully constructed, and the size of the insert is 2500 bp. This study provide the theoretical and material foundation for further study on the function of human KAP-1 gene,