生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2015年
4期
565-569
,共5页
杨艳%朱莹%朱美勤%韦炎龙%倪孟祥%方宏清
楊豔%硃瑩%硃美勤%韋炎龍%倪孟祥%方宏清
양염%주형%주미근%위염룡%예맹상%방굉청
Red同源重组%抗性互补%直接克隆
Red同源重組%抗性互補%直接剋隆
Red동원중조%항성호보%직접극륭
Red homologous recombination%complementary resistance%direct cloning
目的:利用抗性互补策略提高Red重组介导的体内直接克隆效率。方法:将抗性基因(如cat)拆分为互补的两部分Cma、Cmb(部分与Cma同源),这两部分单独均不具有抗性,仅当融合在一起时才有抗性。首先将Cmb定向整合到染色体上拟克隆区域的一侧,然后将含有Cma的线性质粒载体电转到细胞内进行定向克隆。结果:使用该方法,我们成功地将大肠杆菌DH1基因组上约48 kb的大片段DNA克隆到质粒载体上。结论:该方法可用于大片段DNA的功能研究。
目的:利用抗性互補策略提高Red重組介導的體內直接剋隆效率。方法:將抗性基因(如cat)拆分為互補的兩部分Cma、Cmb(部分與Cma同源),這兩部分單獨均不具有抗性,僅噹融閤在一起時纔有抗性。首先將Cmb定嚮整閤到染色體上擬剋隆區域的一側,然後將含有Cma的線性質粒載體電轉到細胞內進行定嚮剋隆。結果:使用該方法,我們成功地將大腸桿菌DH1基因組上約48 kb的大片段DNA剋隆到質粒載體上。結論:該方法可用于大片段DNA的功能研究。
목적:이용항성호보책략제고Red중조개도적체내직접극륭효솔。방법:장항성기인(여cat)탁분위호보적량부분Cma、Cmb(부분여Cma동원),저량부분단독균불구유항성,부당융합재일기시재유항성。수선장Cmb정향정합도염색체상의극륭구역적일측,연후장함유Cma적선성질립재체전전도세포내진행정향극륭。결과:사용해방법,아문성공지장대장간균DH1기인조상약48 kb적대편단DNA극륭도질립재체상。결론:해방법가용우대편단DNA적공능연구。
Objective: To apply a complementary resistance strategy to increase the in vivo direct cloning efficien?cy mediated by Red recombination. Methods: The cat cassette was divided into two parts, Cma and Cmb(has a homologous sequence with Cma), each of them has no resistance separately unless the two parts are fused togeth?er. The Cmb fragment was first integrated into a side of the target cloning region, then a linear Cma-containing plasmid vector was electroporated into the cells to directly capture the region. Results: Based on this strategy, we successfully cloned an about 48 kb DNA fragments from E.coli DH1 chromosome. Conclusion: We can use this method in functional studies of large DNA.