This project will develop methodology for high-throughout identification of cDNAs from large segments of genomic DNA cloned into yeast artificial chromosomes (YACs) and from amplified genomic DNA corresponding to a desired micro-dissected chromosome band. We will use cDNA libraries from human spleen, thymus testes, adrenal, thyroid, cerebral cortex, adult brain and fetal brain. We will explore optimal means of arraying target DNAs in nylon membranes. Hybridization selection will be performed on the gridded target DNA using as probe the cDNA library pool in the presence of quenching reagents which block highly abundant or repetitive sequences. Finally, cDNAs hybridizing to a given target will be analyzed for repetitive sequences and for genes mapped to the original genomic track. A set of clones will also be mapped chromosomally and sequenced. The objective is to generate sequencing and mapping information of novel cDNA clones from several genomic locations simultaneously. Hybridization selection is expedient and economizes on cDNA libraries and on quenching reagents. This project should lead to mapped molecular tools for genome wide applications as well as standard reagents and contract services for identification of essentially all human genes.
