The positional cloning of human disease genes localized by linkage analysis or other genetic methods is often laborious, time-consuming and expensive. We will develop a new methodology that combines subtractive and positional cloning strategies. The starting material is mRNA from paired tissue or cell types that are expected to differ in expression of the target disease gene. Unlike other subtractive methods, however, positional information is incorporated by the direct selection of cDNAs encoded by cosmids, P1 clones, or yeast artificial chromosomes (YACs) within the target genetic region. Subtraction of the two pools of selected cDNAs is then performed using a PCR-based method adapted from "representational difference analysis" of Lisitsyn et al. We will apply this hybrid method, which we call "selection subtractionNational Center for Human Genome Research (NCHGR)
