Novel methods for cloning and engineering genes using the polymerase chain reaction

Abstract

Use of the polymerase chain reaction (PCR) has become increasingly widespread in virtually all aspects of molecular biology. Recently, novel ligation-independent methods have been developed for the cloning of DNA fragments amplified using PCR. Ligation-independent cloning utilizing the enzyme uracil DNA glycosylase (termed UDG cloning) provides an efficient method for gene cloning and recombinant PCR. This technology is now being applied to site-directed mutagenesis, the generation of nested deletions, and the engineering of novel gene constructs. The ease and flexibility of this methodology, combined with PCR amplification, simplify gene cloning and engineering techniques.