Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis

We describe a simple strategy for improving site-specific mutagenesis. We have combined the polymerase chain reaction (PCR) with a phage lambda exonuclease (Exo lambda) treatment to produce mutated fragments larger than 2.5 kb. The applicability of this approach has been proven with two overlapping mutated primers. The procedure has also been made more cost-effective by the use of a single mutated primer, which is referred to as SMP-PCR procedure. The entire procedure of kinasing the primer, amplification by PCR, Exo lambda digestion and second step of PCR can be performed in less than 6 h. We have used this approach to generate a number of mutations in the Salmonella typhimurium hisP gene of the histidine transport operon.