Cloning Polymerase Chain Reaction (PCR) Products: Blunt-End Cloning.

Abstract

The following is an elegant and simple protocol for generating and cloning blunt-ended DNA. Incubation of a ligation reaction in the presence of an excess amount of restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are regenerated when plasmid molecules ligate to themselves. The method requires that ligation of the plasmid to a target DNA molecule destroys the restriction site, so preventing the restriction enzyme from digesting recombinants generated during the ligation reaction. The net effect of constant reclamation of unit-length linear vector molecules is to drive the equilibrium of the ligation reaction strongly in favor of recombinants between vector and insert. The method is efficient because regeneration of vector DNA, ligation, and polishing the termini of PCR-generated fragments of DNA all occur simultaneously in the same reaction mixture.