Cloning human chromosome 9-specific cDNAs by an efficient selection strategy using somatic hybrids

Several disease genes have been mapped to human chromosome 9. To identify candidate genes, we have developed an efficient strategy to select chromosome 9-specific cDNAs. A human fetal brain cDNA phage library was divided into fractions to reduce complexity. The cDNA inserts amplified by vector primers from individual fractions were hybridized in solution to genomic DNA of somatic hybrid-containing chromosome 9. The non-hybridized cDNAs were separated from the genomic DNA and the hybridized cDNAs. These retained cDNAs were then amplified by vector primers and cloned. cDNAs obtained by this approach have been used as probes in Southern hybridization and shown to be positive in both total human and chromosome 9-specific hybrid DNA but not hamster DNA. Preliminary sequence analysis indicates some cDNAs are ESTs with unknown chromosome locations and are now assigned to chromosome 9. A panel of somatic hybrids containing portions of chromosome 9 is being used to determine the regional localization of these cDNAs. Further work for completing the isolation of cDNAs from all fractions of the cDNA library and use of additional cDNA libraries is in progress. We have demonstrated that chromosome-specific cDNAs can be selected by very complex genomic DNAs of somatic hybrids using this protocol,more » which would also be applicable to pools of YAC or cosmid clones. This strategy should increase the efficiency of creating a transcriptional map of chromosome 9 and facilitate the isolation of disease genes as a complementary method to other available techniques.« less