Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites.
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.
Tillett
D
D
School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, Australia.
Neilan
B A
BA
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction / Q000379:methods*