自身引物菌落聚合酶链反应筛选LoxP序列重组阳性克隆

Screening of LoxP-positive recombinant clones by self-primer colony polymerase chain reaction

目的 研究LoxP序列小片段重组阳性克隆的筛选和鉴定方法。方法 采用合成的LoxP为上游引物,载体互补的序列为下游引物,直接以菌落为模板进行聚合酶链反应(PCR),电泳分离出784 bp的条带者为阳性;PCR阳性克隆再用酶切法进行对比鉴定及DNA测序分析确证。结果 用菌落PCR法随机筛选的6个菌落中3个为阳性,随机取1个阳性克隆进行DNA序列分析,证实含有LoxP插入序列。用酶切法对此3个阳性克隆进行鉴定,3个克隆和未进行酶切的对照均出现100 bp以下模糊带,结果不能判断。结论 自身引物菌落:PCR法用于筛选和鉴定LoxP序列重组阳性克隆是一种快速、简便、经济、高效的方法。

Objective To investigate the screening and evaluating methods of positive recombinant clones for small fragments such as LoxP sequence. Methods Synthesized LoxP and vector complementary sequence were used as the upper and lower primer respectively, and colonies were used directly as the templates of polymerase chain reaction (PCR). The presence of 784 bp strap in electrophoresis was seen as positive. The positive recombinant clones screened by PCR were evaluated contrastively by restriction endonuclease digestion and verified by DNA sequence analysis. Results Among the six colonies randomly screened by PCR, three showed positive straps and one was verified by DNA sequence analysis. However, the electrophoresis only showed unclear and clouding straps when the three positive recombinant clones were evaluated by restriction endonuclease digestion. Conclusion Self-primer colony PCR is a high-speed, convenient, economic and effective method for screening and evaluating of positive clones recombinated by small fragments such as LoxP sequence.