Reconstruction of Large Genomic Segments of DNA by Meiotic Recombination Between YACs

The cloning of megabase-size DNA fragments in yeast artificial chromosomes (YACs) creates new opportunities to study, for example, remote cis -acting elements, higher order chromatin structure, and chromosomal folding in coordinating the transcriptional regulation of large genes. Prior to initiating these types of studies, however, it is necessary to develop reliable and reproducible methods that permit the experimental manipulation (reconstructions, deletions, insertions, point mutations) of the large DNA molecules cloned into YACs. Sheer forces limit the ability to alter these large DNA molecules in vitro. Fortunately, the high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae allows these types of modifications to be accomplished in vivo. The purpose of this chapter is to describe a simple method that utilizes meiotic recombination between overlapping YACs to reconstruct a single clone containing an intact gene.