大引物PCR定点突变方法的改进  

Incorporation of site mutations into a target gene with an improved PCR-based megaprimer method

作　　者：张碧乾[1,2] 邓会群[1,2] 宫彩霞[1,2] 杨青[3] 彭建新[1,2] 洪华珠[1,2] 李爱英[1,2] 

机构地区：[1]华中师范大学生命科学学院,湖北武汉430079 [2]华中师范大学教育部农药与化学生物学重点实验室,湖北武汉430079 [3]上海复旦大学药学院,上海200433

出　　处：《湖北大学学报（自然科学版）》2009年第1期79-81,共3页Journal of Hubei University：Natural Science

基　　金：国家自然科学基金(30770036);教育部高等学校博士学科点专项科研基金(20070511004);教育部留学回国人员科研启动基金资助

摘　　要：对大引物PCR定点突变方法进行改进,主要包括:避免在一个PCR反应中同时使用扩增全长基因的常规引物,选择不同载体克隆原始基因和突变基因,通过抗性选择筛选突变子,从而完全避免原始基因的干扰.实验结果证明利用改进后的方法进行基因点突变,可以减少操作步骤,提高突变频率.＂Megaprimer＂ PCR used to introduce site-mutations into a target gene was improved here, including mainly omitting using of a normal primer pair in one reaction for amplification of a full length gene, and choosing different vectors for cloning of wild type and mutant genes, respectively. Therefore, mutants were yielded efficiently by subsequent selection on antibiotics-containing medium, and sequencing analysis showed that interference of wild type genes was completely eliminated by this improved method.

关 键 词：改进的大引物PCR点突变 基因克隆 

分 类 号：Q978[生物学]