Direct hybridization of large-insert genomic clones on high-density gridded cDNA filter arrays.

摘要: A major challenge to positional cloning approaches is the identification of coding sequences within a region interest. Hybridization genomic fragments that represent cloned contig defined in appropriate cDNA libraries theoretically represents direct solution this problem. However, technically difficult and general, success with approach has been limited use small fragments, such as those cosmids phages. Since most physical maps are composed DNA vectors significantly greater insert size capacity, there need develop efficient methods these clones directly hybridization probes. Here we describe highly sensitive protocol for P1-derived artificial chromosomes (PACs; average size, 120 kb) on composite, normalized library comprised 200000 spotted at high density nylon filters. Because sequence information more than 150000 now available public domain, positive results can be rapidly converted without recourse any clone manipulation initial phases project. Using protocols, have able reproducibly detect exons constitute little 0.2% total PAC insert.