[Region-specified PCR-mutagenesis: its application to locate epitopes for anti-RecA protein-monoclonal IgGs ]

Even though various techniques for site-directed mutagenesis have been developed, random mutagenesis is an important and complementary approach to locate functional domains on polypeptides and RNA, and to modify their biochemical and biological properties. Region-specified PCR-mutagenesis is a powerful and simple technique for this purpose. A couple of primers specifies a DNA region to be mutagenized. By use of Taq DNA polymerase and the addition of deoxyinosine-triphosphate (dITP), PCR causes various base-substitutions in the region. By screening of a change in a phenotype of the protein, one can obtain mutants with significant phenotype at 10(-2) from an expressed library of mutagenized DNA.