A random mutagenesis procedure: application to the POL3 gene of Saccharomyces cerevisiae

Abstract To obtain a broad spectrum of mutations in the POL3 gene, we have developed an efficient random mutagenesis procedure. Partially extended, primed, single-stranded templates were used for forced misincorporation of noncomplementary nucleotides, extended to completion and ligated. Linear fragments of the resulting amplified mutagenized library were then used to transform a Saccharomyces cerevisiae strain by the marker replacement technique. This procedure has proven to be very efficient when applied to the C-terminal moiety of POL3 , yielding 24 temperaturesensitive mutants and six extragenic revertants.