Tillett D, Neilan B A
School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, Australia.
Nucleic Acids Res. 1999 Oct 1;27(19):e26. doi: 10.1093/nar/27.19.e26.
We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation-hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This 'enzyme-free cloning' procedure is highly efficient, and is not constrained by the need for the presence of suitable restriction enzyme sites within the plasmid vector. The avoidance of post-amplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps.
我们描述了一种无需进行扩增后酶处理即可克隆PCR产物的简单方法。通过PCR后变性-杂交反应,使用带尾PCR引物组在插入片段和载体上产生互补的交错突出端。单链突出端的设计允许以无连接酶的方式进行定向克隆。这种“无酶克隆”方法效率很高,不受质粒载体中是否存在合适的限制性酶切位点的限制。避免扩增后酶促步骤使该技术快速可靠,无需多个亚克隆步骤。