An enrichment selection for mutants resulting from oligonucleotide-directed mutagenesis of double-stranded DNA

We report here a simple and rapid procedure for enrichment and selection of mutants from oligonucleotide-directed mutagenesis on double-stranded plasmid DNA. Mutagenic oligonucleotides were designed to insert or delete a unique restriction site with silent codon changes. After mutagenesis, plasmid DNA from all resulting colonies was pooled, restricted with the appropriate endonuclease, and the resulting unique form of DNA (linear or circular) was isolated and used for transformation of competent E. coli. These procedures provided an enrichment of mutant plasmid from the 4% obtained by more conventional techniques to greater than 65%.