Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

Abstract

A modified polymerase chain reaction (PCR)-based site-directed mutagenesis method used to splice together different regions of a gene by deleting hundreds of nucleotides of undesired sequences is described. This method was inspired by a PCR-based site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA); the procedure and primer design were modified to enable the method to generate deletions several hundreds of nucleotides in length with an efficiency of 80–100%, and to delete two DNA fragments simultaneously in a single PCR. This method should be useful for deletion of large DNA fragments from a gene.