A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA |
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Authors: | A V Bellini F de Ferra G Grandi |
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Institution: | ENIRICERCHE S.p.A., Department of Molecular Biology, Milan, Italy. |
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Abstract: | 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. |
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