[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.
Shibata
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Laboratory of Cellular & Molecular Biology, Institute of Physical and Chemical Research (RIKEN).
D000911:Antibodies, Monoclonal; D004259:DNA-Directed DNA Polymerase; D000939:Epitopes / Q000032:analysis*; D007074:Immunoglobulin G; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase