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A major challenge to positional cloning approaches is the identification of coding sequences within a region of interest. Hybridization of genomic fragments that represent a cloned contig of a defined genomic region in appropriate cDNA libraries theoretically represents a direct solution to this problem. However, this is technically difficult and in general, success with this approach has been limited to the use of small fragments, such as those cloned in cosmids and phages. Since most physical maps are composed of genomic DNA cloned in vectors with significantly greater insert size capacity, there is a need to develop efficient methods to use these clones directly as hybridization probes. Here we describe a highly sensitive protocol for hybridization of P1-derived artificial chromosomes (PACs; average insert size, 120 kb) on a composite, normalized cDNA library comprised of 2 clones spotted at high density on nylon filters. Because limited sequence information on more than 150000 of these clones is now available in the public domain, positive hybridization results can be rapidly converted to cDNA sequence information without recourse to any clone manipulation in the initial phases of a project. Using these protocols, we have been able to reproducibly detect coding exons that constitute as little as 0.2% of the total PAC insert.

Direct Hybridization of Large-Insert Genomic Clones on High-Density Gridded cDNA Filter Arrays