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

Abstract:

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.

Nucleic Acids Research.1999:27(19):e26

School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, Australia.

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