EASY 克隆 - 北京全式金生物(TransGen Biotech) 官方主页.PDF

EASY 克隆“1+1” ——5分钟快速、高效克隆 北京全式金生物技术有限公司 分子克隆的传统方法 Topoisomerase (EASY)克隆原理 EASY 克隆策略 EASY 克隆的几个细节 无缝拼接基因克隆 Some slides are copied from several web sites. Thanks to all of these unknown contributors! 分子克隆的传统方法 分子克隆的传统方法 连接和环化 • 常规的连接是一个三分子共同反应的过程。 • TA克隆中粘性末端仅有一个碱基，因此较高 的DNA浓度和较高T4连接酶浓度是获得较高 连接效率的前提。 • 环化过程恰恰与连接过程相反，过高的DNA浓度和T4连接酶浓度会增加分子间 相互作用促进串连体(concatemer)形成。 • 通常情况下, 克隆效率（连接产物转化效率，而不是克隆阳性率）低于0.1% 。 Topoisomerase (EASY)克隆原理 拓扑异构酶的功能 Vaccina Topoisomerase as Cloning Tool • Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. S Shuman and J Prescott . J. Biol. Chem., Vol. 265, Issue 29, 17826-17836, Oct, 1990 • Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro. S Shuman . J. Biol. Chem., Vol. 266, Issue 17, 11372-11379, Jun, 1991 • Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. S Shuman J. Biol. Chem., Vol. 269, Issue 51, 32678-32684, 12, 1994 EASY 克隆原理 连接反应成为一 个双分子作用的 过程 TOPO载体反应动态平衡 Mary R. Stahley and James T. Stivers Biochemistry.2010 April 6: 49(13):2786-2795 Reversible DNA cleavage and ligation by topoisomerase IB and DNA strand exchange. The native strand (black) is cleaved during the first transesterification reaction, and can dissociate from the Topo-DNA covalent complex. Another DNA strand with a 5-OH (red) can associate and attack the DNA-protein phosphotyrosyl bond, resulting in DNA rearrangements. 传统TA载体和TOPO载体稳定性、自连率的比较