Department of Veterinary Microbiology, Veterinary Medical Research Institute, 1802 University Boulevard, Iowa State University, Ames, IA 50011, USA.
Plasmid. 2011 Sep;66(3):180-5. doi: 10.1016/j.plasmid.2011.07.007. Epub 2011 Aug 10.
Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.
已经开发出了几种无需连接的克隆方法，这些方法在高效构建重组质粒的同时，最大限度地减少了克隆伪迹。在这里，我们报告了新的质粒载体，这些载体使用切口内切酶 Nt.BspQI 生成扩展的单链尾巴，用于直接克隆 PCR 产物。这些载体包括 pLacCOs1，一种 ColE1 衍生的质粒，赋予氨苄青霉素抗性，允许轻松构建 lacZ 翻译融合物，以及 pKanCOs1，一种 pSC101 衍生的克隆载体，赋予卡那霉素抗性，用于从基因组 DNA 以及基于氨苄青霉素的质粒中克隆 PCR 扩增子。我们已经成功地使用这些质粒定向克隆和表征了具有温度调节表达的细菌启动子，以及克隆各种 PCR 产物。在所有情况下，都以高效率和最少的操作生成了具有正确构型的构建体。这些克隆载体也可以很容易地进行修改，以纳入其他报告基因或表达表位标记的基因产物。
