Triton X-100在单菌落PCR技术中的优化作用  

Study on Improvement Effect of Triton X-100 for Individual Bacterial Colony PCR Method

作　　者：张晓莉[1] 冯莹颖[1] 张强[1] 罗勤[1] 蒋苹[2] 钱悦[2] 

机构地区：[1]华中师范大学生命科学学院遗传调控与整合生物学湖北省重点实验室,湖北武汉430079 [2]华中科技大学附属协和医院皮肤科,湖北武汉430022

出　　处：《化学与生物工程》2009年第6期82-85,共4页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(30500025和30600535)

摘　　要：在利用单菌落PCR法直接筛选含有外源基因的重组质粒以及发生同源重组的重组子时,通过0.1%TritonX-100处理模板对PCR技术进行优化。以大肠杆菌DH5α、单核细胞增生李斯特菌的重组克隆为例,单菌落经Triton X-100处理后再进行PCR,琼脂糖凝胶电泳图谱的条带更清晰、杂带减少;克隆鉴定的阳性率明显提高,且在15μL的PCR反应混合液体系中的扩增效果更明显。对单核细胞增生李斯特菌的检测方法可以推广适用于多种革兰氏阳性菌。The individual bacterial colony PCR method was used to screen the recombinant plasmids containing foreign genes and homologous recombinants directly. The clones were cultured and selected from E. coli DHSa, Listeria monocytogenes respectively. The methods were improved by treating PCR templates with 0.1 % Triton X-100. From the agarose gel electrophorograms, it was found that the positive identification rate of screening could be increased dominantly through treatment of template by Triton X-100. This study suggested that the individual bacterial colony PCR method could be improved by treatment of PCR templates with 0.1% Triton X-100, and a system of 15 μL PCR mix would be the best choice. The special method for Grampositive bacteria could be applied to the promotion of a variety of pathogens.

关 键 词：TRITONX-100 单菌落PCR 重组克隆 筛选 鉴定 

分 类 号：Q78[生物学—分子生物学]