Cloning and Assembly of PCR Products Using Modified Primers and DNA Repair Enzymes

Abstract

We present a method for the creation of ligatable 3′overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.