Using a modified TA cloning method to create entry clones
Using a modified TA cloning method to create entry clones
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DOI：
10.1016/j.ab.2006.08.015
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发表时间：
2006-11-01
影响因子：
2.9
通讯作者：
Wang, Xue-Chen
中科院分区：
文献类型：
--
作者：
Chen, Qi-Jun;Zhou, Hai-Meng;Wang, Xue-Chen
我们描述了一种非商业化的替代方法来创建兼容的所有类型的目的载体的基础上改进的TA克隆方法的入口克隆。为了产生Gateway T载体，我们首先构建了分别命名为pGWG和pGWC的庆大霉素和氯霉素抗性进入载体。每个进入载体含有两侧为attL位点的AhdI盒，每个AhdI盒含有两个由ccdB杀伤基因间隔的AhdI限制性酶位点，其对大多数大肠杆菌菌株是致命的。Gateway T载体可以通过用AhdI酶或其异构体简单消化这些进入载体来制备。使用ccdB基因作为阴性选择标记是对常规TA克隆的重要改进，因为它消除了基于α互补的蓝/白色筛选的必要性。另一个重要的改进，我们已经实施的是零售的T载体使用Taq聚合酶和dTTP，以提高克隆效率。总之，这些改进使TA克隆能够充分发挥其潜力。使用通过这种改进的方法制备的Gateway T载体，可以简单、有效和廉价地产生PCR产物或限制酶片段的入口克隆，同时引入更大的相容性。(c)2006年爱思唯尔公司All rights reserved.
We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on alpha-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility. (c) 2006 Elsevier Inc. All rights reserved.