Building Larger YACs by Recombination

AbstractDespite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact, but are represented as a collection of overlapping clones or contigs. Fortunately, the relatively high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae can be used to reconstruct intact genes within a single clone by splicing together overlapping DNA segments. This unit describes two protocols for carrying out such homologous recombination; one relies on the meiotic phase of the yeast cycle, while the other utilizes the mitotic phase of the yeast life cycle.Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact.