Enzyme-free cloning: A rapid method to clone PCR products independent of vector restriction enzyme sites

摘要

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 postamplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps.