YAC subclone contig assembly by serial interspersed repetitive sequence (IRS)-PCR product hybridizations.

Yeast artificial chromosomes (YACs) are presently the most effective means of cloning large contiguous regions of genomic DNA (1). However, performing a detailed molecular analysis of YAC clone insert DNA is constrained by inherent physical and technical limitations. Typically, an extended analysis of YAC DNA is facilitated by subcloning YAC insert DNA into lambda bacteriophage or cosmid vectors and constructing a contig map (2-4). In the past, a variety of strategies and techniques have been used to construct a contig map of YAC subclones; these have included the use of species-specific repetitive DNA to perform hybridization-based sequence fingerprint analyses (2) and the use of subclone-specific vector-Alu PCR products (3) and RNA end-clone fragments (4) to identify partially overlapping subclones. Each of these techniques, however, requires that individual subclones be isolated prior to analysis. Since, to ensure adequate representation, it is typical to construct a cosmid or bacteriophage library representing 5 to 10 genome equivalents ofYAC insert DNA (3, 4), these techniques require the isolation and analysis of numerous individually purified clones. The construction of a contig map would be expedited by delaying the isolation of individual clones until a minimal tile contig is deduced. Here we report on the use of interspersed repetitive sequence (IRS)-PCR products derived from YAC insert DNA to construct a contig map of bacteriophage subclones prior to the isolation of individual purified clones. To simultaneously identify phage clones containing an IRS-PCR product and localize the product to a specific restriction fragment within the YAC insert, each IRS-PCR product was concurrently hybridized both to filters containing phage plaques plated on numbered grids and blots containing digested YAC DNA. High molecular weight yeast DNA was isolated by using the