一种简单高效的酵母单菌落PCR方法  

A Simple and Effective Method for Yeast Colony PCR

作　　者：陈忠翔[1] 房志家[1] 陈婷[1] 赵敏[1] 黄志伟[1] 

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《生物技术通讯》2013年第2期225-229,共5页Letters in Biotechnology

基　　金：国家自然科学基金(31100549);中央高校基本科研业务费专项资金(2011D10512);上海浦江基金(11PJ1400100)

摘　　要：目的:为快速简便地挑选出酿酒酵母重组克隆,探索建立一种经济、直接、高效的酵母单菌落PCR方法。方法:以Leu2MX6基因同源重组或重组质粒转化得到的酵母突变菌为材料,分别采用传统的提取基因组或质粒的方法、煮沸法及化学试剂处理法等制备PCR模板进行重组克隆鉴定,并对6种PCR模板制备方法的效果进行比较与分析;对加热提取法进行优化并进行重组子的提取和验证。结果与结论:直接以1 mm2单克隆菌株95℃处理5 min后的酵母菌落水悬浮液为模板进行单菌落PCR,是一种简单高效的酵母重组克隆鉴定方法。该方法能弥补传统方法的不足,且简便快速、结果稳定,可作为筛选和鉴定阳性克隆的有效手段。同时,这种单菌落PCR法也可应用于重组毕赤酵母的阳性克隆筛选。Objective： In order to pick out the recombinant clones of Saccharomyces cerevisiae quickly and easily, an economic, simple and effective yeast single colony PCR method was developed. Methods： Several DNA tem- plate preparation methods have been used from transformation, including the traditional genome or plasmid extrac- tion, directly boiling and/or treating with a variety of chemical reagents. After PCR amplification and detection by agarose gel electrophoresis, the effects of DNA template preparation on PCR were evaluated. Results ＆ Conclu- sion： A novel method for template DNA preparation, treating the 1 mm2 monoclonal strain with 95~C for 5 min di- rectly, had the advantage of simpleness and high-efficiency. This novel method can be used as an effective means of screening and identification of positive recombinant clones of S.cerevisiae. Meanwhile, it also could be applied to screen the positive clones from recombinant of Pichia pastoris.

关 键 词：酵母 菌落PCR 重组克隆 筛选 

分 类 号：Q78[生物学—分子生物学]