酵母质粒PCR扩增及酶切在酵母双杂交中的运用  

THE APPLICATION OF PCR AMPLIFICATION DIGESTED WITH ENDONUCLEASE OF YEAST PLASMID IN YEAST TWO-HYBRID EXPERIMENTS

作　　者：孙玉宁[1] 李春荣[2] 靳更林[3] 

机构地区：[1]宁夏医学院生物化学教研室,宁夏银川750004 [2]承德医学院 [3]北京大学临床肿瘤学院生化室

出　　处：《承德医学院学报》2005年第3期190-192,共3页Journal of Chengde Medical University

摘　　要：目的:减少对初步筛选获得的阳性克隆进一步鉴定的工作量,防止阳性克隆的丢失。方法:提取酵母双杂交筛选cDNA表达文库获得的阳性克隆酵母细胞质粒,以文库载体插入片段两端特异序列为引物,PCR扩增插入片段,限制性核酸内切酶酶切扩增产物,琼脂糖凝胶电泳,根据带型对阳性克隆进行分类。结果:将初步获得的107株阳性克隆,分为24类,在PCR扩增过程中发现个别克隆含有两种以上的文库质粒。结论:酵母质粒PCR扩增,酶切分类的方法,不仅可以大大减轻阳性克隆进一步鉴定的工作量,避免不必要的浪费,而且还可以避免阳性克隆的丢失。Objective： To decrease the further identification workload and to avoid the lost of positive clones. Methods： After isolating plasmid DNA from each positive yeast clone, cDNA library inserts were amplified by PCR using special sequences on library vector as primer. The PCR products were characterized by digesting with a frequent-cutter restriction enzyme, then the products were performed by agarose gel electrophoresis. Positive clones were classified according to the clone＇s characters. Results： 107 primary positive clones were notarized into 24 different positive clones, and few positive clones were found to have two kinds of plasmid by PCR. Conclusions： The further identification workload was decreased greatly for positive clones and the lost of positive clones were avoided by applying this method.

关 键 词：酵母双杂交 阳性克隆 PCR 限制性核酸内切酶 

分 类 号：Q503[生物学—生物化学]