METHOD OF ORDERING CHROMOSOME-SPECIFIC OVERLAPPING CLONES

IN THE PROPOSED RESEARCH WE WILL DEVELOP METHODOLOGIES AND PRODUCTS FOR MAPPING VERY LARGE PIECES OF DNA BY DETERMININGTHE OVERLAPPING RELATIONSHIPS BETWEEN CLONES DERIVED FROM A PARTIAL DIGEST OF THE DNA. THE METHOD WILL CREATE THE POSSIBILITY OF ORDERING END-TO-END A LARGE NUMBER OF PARTIAL-DIGEST COSMID CLONES DERIVED FROM SEGMENTS OF DNA ASLARGE AS AN ENTIRE HUMAN CHROMOSOME. THE PROPOSED METHOD INVOLVES CROSS-HYBRIDIZATION OF THE INSERTS FROM A LARGE NUMBER OF COSMID CLONES TO EACH OTHER INDEPENDENTLY IN ALL COMBINATIONS BY APPLYING CLONED DNA OR PROBES GENERATED FROMTHE CLONES TO SHORT PIECES OF THREAD. THESE THREADS ARE SUBSEQUENTLY WOVEN IN ORDER INTO CLOTH. THE INTERSECTION POINTS OF THE THREADS CROSSING EACH OTHER IN THE CLOTH ALLOWS FOR DISCRETE HYBRIDIZATION OF ALL CLONED DNAS SEPARATELY AND INDEPENDENTLY TO ALL PROBES GENERATED FROM THE SAME SET OF CLONES AND, THEREFORE, ALLOWS FOR SIMULTANEOUS DETERMINATION OF THE OVERLAP BETWEEN ALL CLONESIN THE SET. THE PROPOSED METHOD, IF IMPLEMENTED, WOULD BE VERY POWERFUL FOR ITS INTENDED PURPOSE AND WOULD REDUCE THE CLONE ORDERING TASK INTO A SIMPLIFIED, STANDARDIZED, AND EASILY REPEATABLE PROTOCOL. IN PHASE I WORK, WE PROPOSE TO TRY THE METHOD ON THE E. COLI GENOME SO THAT THE PROCEDURE CAN BE VERIFIED AND OPTIMIZED BEFORE ATTEMPTING AN ENTIRE HUMAN CHROMOSOME OVERLAPPING CLONE DETERMINATION FROM A PREEXISTING FLOW-SORTED, CHROMOSOME-SPECIFIC LIBRARY.