Bellini A V, de Ferra F, Grandi G
ENIRICERCHE S.p.A., Department of Molecular Biology, Milan, Italy.
Gene. 1988 Sep 30;69(2):325-30. doi: 10.1016/0378-1119(88)90442-8.
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.
