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.
Bellini
A V
AV
ENIRICERCHE S.p.A., Department of Molecular Biology, Milan, Italy.
de Ferra
F
F
Grandi
G
G
D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D010566:Virulence Factors, Bordetella / Q000235:genetics
D010566:Virulence Factors, Bordetella