生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2013年
2期
225-229
,共5页
陈忠翔%房志家%陈婷%赵敏%黄志伟
陳忠翔%房誌傢%陳婷%趙敏%黃誌偉
진충상%방지가%진정%조민%황지위
酵母%菌落 PCR%重组克隆%筛选
酵母%菌落 PCR%重組剋隆%篩選
효모%균락 PCR%중조극륭%사선
yeast%colony PCR%recombinant clone%screening
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基同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 法也可应用重组毕赤酵母的阳性克隆筛选.
目的:為快速簡便地挑選齣釀酒酵母重組剋隆,探索建立一種經濟、直接、高效的酵母單菌落 PCR 方法.方法:以 Leu2MX6基同重組或重組質粒轉化得到的酵母突變菌為材料,分彆採用傳統的提取基組或質粒的方法、煮沸法及化學試劑處理法等製備 PCR 模闆進行重組剋隆鑒定,併對6種 PCR 模闆製備方法的效果進行比較與分析;對加熱提取法進行優化併進行重組子的提取和驗證.結果與結論:直接以1 mm2單剋隆菌株95℃處理5 min 後的酵母菌落水懸浮液為模闆進行單菌落 PCR,是一種簡單高效的酵母重組剋隆鑒定方法.該方法能瀰補傳統方法的不足,且簡便快速、結果穩定,可作為篩選和鑒定暘性剋隆的有效手段.同時,這種單菌落 PCR 法也可應用重組畢赤酵母的暘性剋隆篩選.
목적:위쾌속간편지도선출양주효모중조극륭,탐색건립일충경제、직접、고효적효모단균락 PCR 방법.방법:이 Leu2MX6기동중조혹중조질립전화득도적효모돌변균위재료,분별채용전통적제취기조혹질립적방법、자비법급화학시제처리법등제비 PCR 모판진행중조극륭감정,병대6충 PCR 모판제비방법적효과진행비교여분석;대가열제취법진행우화병진행중조자적제취화험증.결과여결론:직접이1 mm2단극륭균주95℃처리5 min 후적효모균낙수현부액위모판진행단균락 PCR,시일충간단고효적효모중조극륭감정방법.해방법능미보전통방법적불족,차간편쾌속、결과은정,가작위사선화감정양성극륭적유효수단.동시,저충단균락 PCR 법야가응용중조필적효모적양성극륭사선.
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℃ 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.
