ABSTRACT
With the development of techniques in molecular biology, isolation and study of a single gene is now possible and mutagenesis has been refined. This chapter describes some basics in vitro mutagenesis strategies, each of which is used for creating specific types of mutations in cloned DNA sequences. The strategies recently developed for in vitro site-directed mutagenesis allow one to create virtually any change at will and this—in principle—enables us to correlate any piece of gene sequence information with gene regulation and product formation. The chapter illustrates how a plasmid insert can be mutated by restriction enzyme mediated deletion mutagenesis using compatible restriction enzyme mediated termini, or by digesting linearized plasmid DNA with exonucleases followed by treatment with Klenow DNA polymerase. A key strategy in the oligonucleotide-directed mutagenesis method is to increase the relative number of mutated plasmids in the pooled population.
