Improvement Of Oligonucleotide-Directed Site-Specific Mutagenesis Using Double-Stranded Plasmid DNA

Abstract

Conditions which gave a high yield of oligonucleotide-directed site specific mutagenesis were obtained with use of the lpp gene of Escherichia coli cloned in the versatile expression plasmid vector, pIN-II. This plasmid (5.6-kb) was digested by PstI followed by exonuclease (Klenow fragment of DNA polymerase I) to remove a portion of the gene for β-lactamase. Another sample was digested by Xbal which cuts at a unique site in the pIN-II vector, and EcoRI to remove the portion of the lpp gene to be mutagenized. Both fragments were mixed with a synthetic oligonucleotide (the mutagen) and the mixture was denatured at 100°C for 3 min, followed by gradual cooling to reanneal. The mixture of renatured DNA was treated with the Klenow fragment of DNA polymerase I plus deoxynucleotide triphosphates in the presence of T4 ligase, and ampicillin-resistant transformants were isolated. In the case of a single base substitution, the yield of the desired mutant was as high as 13%. The present method can be applied to any gene cloned in a plasmid vector and is more convenient and simpler than the method using an M13 phage vector, since the mutated gene can be expressed directly from the plasmid vector used for the mutagenesis.