A random mutagenesis procedure: application to the POL3 gene of Saccharomyces cerevisiae.
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 non-complementary 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 temperature-sensitive mutants and six extragenic revertants.
Simon
M
M
Institut Curie, Section de Biologie, Centre Universitaire, Orsay, France.
Giot
L
L
Faye
G
G
D001482:Base Composition; D004258:DNA Polymerase III; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005800:Genes, Fungal; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013696:Temperature
D004277:DNA, Single-Stranded; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase