Protocol

PCR Mutagenesis by Overlap Extension and Gene SOE

This protocol was adapted from "Mutagenesis and Synthesis of Novel Recombinant Genes Using PCR," Chapter 32, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Mutagenesis by PCR is accomplished by incorporating desired genetic changes into custom-made primers used in amplification reactions. Because these mutagenizing primers have terminal complementarity, two separate DNA fragments amplified from a target gene can be fused into a single product by primer extension without relying on restriction endonuclease sites or ligation reactions. Briefly, mutagenesis is achieved by performing PCR with specially designed oligonucleotide primers that include the desired substitutions, insertions, or deletions in their sequence. The two overlapping fragments are fused together in a subsequent extension reaction. The inclusion of outside primers in the extension reaction amplifies the fused product by PCR. Theoretically, the primers can be moved anywhere along the targeted gene to introduce mutations. This method can be exploited further by using DNA fragments from different sources. Such gene splicing by overlap extension (SOE) can be used to rapidly produce chimeras. A limitation of SOE is the difficulty of manipulating large DNA segments (i.e., >1-2 kb). To circumvent this, a cassette system can be targeted, modified by SOE, and reinserted using restriction endonuclease sites designed into the cassette structure. This approach also allows easy shuffling or replacement of gene segments.