NEW CONSTRUCTS AND STRATEGIES FOR EFFICIENT PCR-BASED GENE MANIPULATIONS IN YEAST

Gene disruption and tagging can be achieved by homologous recombinatio n in the yeast genome. Several PCR-based methods have been described t owards this end. However these strategies are often limited in their a pplications and/or their efficiencies and may be technically demanding . Here we describe two plasmids for C-terminal tagging of proteins wit h the Ige binding domain of the Staphyloccocus aureus protein A. We al so present simple and reliable strategies based on PCR to promote effi cient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be use d for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for conse cutive introduction of various constructs into a single yeast strain. (C) 1998 John Wiley & Sons, Ltd.