Restriction enzyme treatment/ligation independent cloning using caged primers for PCR.

Abstract

We report here a new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment. By using caged primers in PCR, unnatural sticky-ends of any length and sequence are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in desired arrangement, tightly enough to be repaired and ligated in competent cells. We have successfully constructed a recombinant vector based on pBR322 and coding GFP gene by applying this simple and effective system.