Department of Microbiology & Immunology, College of Physicians & Surgeons, Columbia University, New York, NY 10032, USA.
FEMS Microbiol Lett. 2012 Dec;337(2):97-103. doi: 10.1111/1574-6968.12016. Epub 2012 Oct 22.
Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically, recombineering uses long PCR primers (c. 65 bases), each of which contains a small region of target homology (c. 45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤ 35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce noncompatible ends. We have used this method to change the bla gene of pACYC177 to aadA, to add the MCS-lacZα region from pBBR1MCS to IncQ plasmid vectors, and to make an oriT(IncP) -aacC1 cassette and add it to a plasmid.
基因重组是一种强大的 DNA 操作方法。它具有优于限制性内切酶方法的优点，通常速度较快。通常，基因重组使用长 PCR 引物（约 65 个碱基），每个引物都包含一小段目标同源序列（约 45 个碱基）。我们开发了一种简单的策略，尽管速度稍慢，但可以使用≤35 个碱基的引物进行所有步骤的基因重组。同源序列的长度可以达到数百个碱基对，以增加获得所需克隆的机会和/或允许基于噬菌体的基因重组在某些非大肠杆菌细菌中进行。该方法使用克隆技术构建用于产生基因重组底物的模板。由于模板是由克隆的 DNA 片段制成的，因此可以轻松更改片段（包括同源区域的片段）。在模板质粒的构建过程中，通过用产生不兼容末端的两种限制性内切酶克隆每个片段，可以显著减少没有插入物的载体产生的潜在背景转化体。我们已经使用这种方法将 pACYC177 的 bla 基因改为 aadA，将 pBBR1MCS 的 MCS-lacZα 区域添加到 IncQ 质粒载体中，并制作 oriT(IncP)-aacC1 盒并将其添加到质粒中。
