Introduction of point mutations into cloned genes

Publisher Summary The genetic analysis of essential genes still relies largely on the generation of point mutations in those genes for an alternative approach, exploiting repressible promoters and ubiquitin destabilization of the gene product. This chapter discusses briefly the principle of the plasmid shuffle technique, which is critical to the generation and analysis of point mutants in essential genes. The chapter also discusses the two approaches to in vitro generalized mutagenesis of cloned yeast genes and describes several methods for the introduction of mutations into a clonable DNA fragment. Where appropriate, methods for introduction of the mutagenized fragment into the full-length yeast gene are also described in the chapter. There are many available techniques for introducing sequence changes into cloned DNAs