Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast

YAC cloning technology has contributed significantly to physical mapping of genomes and holds enormous potential for identification and functional analysis of genes on large DNA segments. This potential is greatly enhanced by the ability to use homologous recombination-based methodologies to restructure or manipulate exogenous DNA in the yeast host. Three methods are described for manipulation of YAC DNA. “Recombinationally targeted YAC cloning” involves the capture of large DNA segments by recombination in vivo between the YAC vector arms and a target DNA segment. “Two-step gene replacement” exploits targeted homologous recombination to replace a selected segment within a YAC with mutant or exogenous sequences. “YAC transfer by kar1 mating” allows rapid movement of YACs under study into yeast genetic backgrounds that facilitate homologous recombination-based manipulation. These three methods taken together will aid significantly in the construction of physical genome maps and provide powerful genetic tools for the study of genome function.