Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease.
Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease.
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DOI：
10.1016/j.plasmid.2011.07.007
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发表时间：
2011-09
期刊：
影响因子：
2.6
通讯作者：
Phillips GJ
中科院分区：
文献类型：
--
作者：
Oster CJ;Phillips GJ
已经开发了几种不依赖于连接的克隆方法，这些方法为重组质粒的构建提供了优势，包括高效率，同时最大限度地减少克隆假象。在这里，我们报告了新的质粒载体，该载体使用切口核酸内切酶 Nt.BspQI 生成延伸的单链尾，用于直接克隆 PCR 产物。这些载体包括 pLacCOs1（一种赋予氨苄青霉素抗性的 ColE1 衍生质粒，可轻松构建 lacZ 翻译融合体）和 pKanCOs1（一种赋予卡那霉素抗性的 pSC101 衍生克隆载体），用于从基因组 DNA 以及基于氨苄青霉素的质粒克隆 PCR 扩增子。我们已经成功地使用这些质粒来定向克隆和表征表现出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配置的构建体。克隆载体还可以很容易地进行修饰，以掺入额外的报告基因或表达表位标记的基因产物。
Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids, including 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.
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