A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA

This paper describes a new method for site-directed mutagenesis which allows mutations by deletion, insertion or substitution of large fragments of DNA with more than 50% efficiency and does not require subcloning in a single-stranded (ss) DNA vehicle. The site of mutagenesis is removed from a linearized plasmid DNA by BAL 31 digestion, ss DNA regions are generated by limited exonuclease treatment and the mutated target site is reconstituted by annealing of the plasmid DNA to a 35-70 nucleotide long mutated ss oligodeoxynucleotide containing the desired mutation. The circularized plasmid is finally used to transform directly Escherichia coli competent cells.