DIRECT HYBRIDIZATION OF LARGE-INSERT GENOMIC CLONES ON HIGH-DENSITY GRIDDED CDNA FILTER ARRAYS

A major challenge to positional cloning approaches is the identificati on of coding sequences within a region of interest. Hybridization of g enomic fragments that represent a cloned contig of a defined genomic r egion on appropriate cDNA libraries theoretically represents a direct solution to this problem. However, this is technically difficult and i n general, success with this approach has been limited to the use of s mall fragments, such as those cloned in cosmids and phages. Since most physical maps are composed of genomic DNA cloned in vectors with sign ificantly greater insert size capacity, there is a need to develop eff icient methods to use these clones directly as hybridization probes. H ere we decribe a highly sensitive protocol for hybridization of P1-der ived artificial chromosomes (PACs; average insert size, 120 kb) on a c omposite, normalized cDNA library comprised of 200 000 clones spotted at high density on nylon filters. Because limited sequence information on more than 150 000 of these clones is now available in the public d omain, positive hybridization results can be rapidly converted to cDNA sequence information without recourse to any clone manipulation in th e initial phases of a project. Using these protocols, we have been abl e to reproducibly detect coding exons that constitute as little as 0.2 % of the total PAC insert.