DEVELOPMENT OF SYSTEMS FOR RAPID CHROMOSOME MAPPING

WE WILL DEVELOP SIMPLE AND VERSATILE SYSTEMS TO MAP MAMMALIAN GENES TO INDIVIDUAL CHROMOSOME AND SUBCHROMOSOME BANDS BY APPLICATION OF THE POLYMERASE CHAIN REACTION (PCR) TECHNIQUE. AMPLIFICAATION IS PERFORMED BY UTILIZING A UNIVERSAL PCR TEMPLATE-ADAPTOR AND PRIMER (CASSETTE) WHICH IS INSERTED RANDOMLY INTO THE CHROMOSOMAL DNA BY LIGATION TO COMBINATIONS OF RESTRICTION ENZYME DIGESTS. A PERMANENT COLLECTION OF THE AMPLIFIED CHROMOSOMAL DNA WILL THEN BE ESTABLISHED BY CLONING INTO A PLASMID VECTOR. WITH THIS SYSTEM CHROMOSOME MAPPING CAN THEN BE PERFORMED IN SEVERAL WAYS; (1) BY HYBRIDIZATION OF CDNA OR GENE FRAGMENT PROVES TO AMPLIFIED CHROMOSOMAL DNA IMMOBILIZED ON HYBRIDIZATION MEMBRANES, (2) BY PCR ANALYSIS TO COLLECTIONS OF THE CLONED CHROMOSOMAL DNA WITH GENE SPECIFIC PRIMER SETS, AND (3) BY USE OF THE CLONED DNA AS CHROMOSOME SPECIFIIC HYBRIDIZATION PROBES. IN PHASE I WE WILL EVALUATE THE FEASIBILITY OF THIS APPROACH USING PCR AMPLIFIED WHOLE GEMONIC DNA. THIS WILL BE FOLLOWED BY AMPLIFICATION OF WHOLE METAPHASE CELL CHROMOSOME DISSECTIONS TO OPTIMIZE THE MANIPULATION AND PROCESSING OF CHROMOSOMES. PHASE II WILL EXTEND THE METHODOLOGY TO DISSECTED INDIVIDUAL CHROMOSOMES AND CONSTRUCT LIBRARIES FROM ALL 23 HUMAN CHROMOSOMES, AS WELL AS SUBCHROMOSOMAL BANDS IN REGIONS OF PARTICULAR INTEREST.