This proposal describes the application of a new technology, termed """"""""preparative in-situ hybridizational (Prep.-I.S.H.) for generating cDNA libraries that map to defined legions of the human genome Prep.-I.S.H. is a combination of two established technologies, in-situ hybridization to metaphase chromosomes, and chromosome microdissection. Briefly, the procedure calls for hybridization of an uncloned cDNA library on the ends with primer binding sites, to metaphase chromosome spreads. The cytogenetically defined legion of interest is then microdissected, and the cDNAs that are bound to the dissected legion are then amplified using the PCR. By specifying the initial source of the cDNA probe library the method can be tailored so that tissue-specific, developmental-stage specific and chromosome legion-specific cDNAs can be generated. This technology will increase the speed of attaining the goals of the NCHGR by allowing certain regions of the genome to be targeted, as well as providing a method for isolating coding regions without the implication of inter and intra-genic DNA sequences. We propose to apply the Prep.-I.S.H. technology, when optimized, to generate legion-specific human chromosome cDNA libraries. The commercialization of such libraries will provide a valuable user friendly and cost effective resource for human genome studies.
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