Abstract
A rapid method directed by exonuclease III and an oligomer for site-specific mut-agenesis using double-stranded plasmid DNA has been developed. In this method a unique non-3’-protruding restriction site must be in close proximity to the functional domain of the target gene cloned in a plasmid. Piasmid linearized at such a site was subjected to limited exonulease III digestion to generate two single-stretches from both open termini. After an annealing step with a mutagenic oligomer and repair synthesis, the duplex DNA was circularized by T4 DNA ligase. Following transforma-tion into E. coli cells, mutants were screened by phenotypic selection and hypbridiza-tion using 32P-labeled mutagenic oligomer as a probe. Gene mutation was furher con-firmed by restriction assay and sequence analysis. Under these conditions, a net mu-tant yield of more than 8% was obtained.
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