Simple approach for oligonucleotide-directed mutagenesis of any double-stranded circular DNA

AbstractA new site-directed method for introducing mutations into any region of plasmid vector close to the unique restriction site is described. It is based on the use of 5′-phosphorylated mutagenic and nonphosphorylated auxiliary oligonucleotides and a specific combination of enzymatic procedures including ‘nick-translation’ as a key step. The method efficiency was demonstrated by constructing the deletion-insertion mutation wich creates the consensus Pribnow box in a promoter-testing plasmid. The yield of the target mutation was up to 85–95%