Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast.

摘要: A valuable weapon in the arsenal available to yeast geneticists is ability introduce specific mutations into genome. In particular, methods have been developed deletions genome using PCR fragments. These are highly efficient because they do not require cloning plasmids. We modified existing method for introducing (S. cerevisiae) fragments order target point this describe two PCR-based directing such that final product contains no other disruptions. first method, site-specific genomic (SSG) mutagenesis, a mutation targeted second random domain-localized (RDL) introduced at within domain of gene. Both sequential transformations, transformation integrates URA3 marker locus, and replaces with fragment containing one or few mutations. This synthesized primer (SSG mutagenesis) by error-prone (RDL mutagenesis). SSG proximal site incorporated higher frequencies than distal mutations, however can be efficiently distances least 500 bp from insertion. RDL ensure incorporation occurs approximately equal throughout region, region deleted same time integrated. mutagenesis allow easily without disrupting native locus.