AUTOMATED LARGE SCALE ANALYSIS OF HUMAN COSMID LIBRARIES

THE OBJECTIVE OF THE PROPOSED RESEARCH PLAN IS TO DEVELOP METHODS TO RAPIDLY SCREEN, ISOLATE, AND CHARACTERIZE COSMID CLONES FROM HUMAN DNA LIBRARIES. IN PHASE I WE WILL ESTABLISH PROCEDURES FOR USING A ROBOTIC WORKSTATION TO AUTOMATE THE SCREENING OF A COSMID LIBRARY PREPARED FROM A SOMATIC CELL HYBRID THAT CONTAINS HUMAN CHROMOSOME 21. WE WILL ALSO OPTIMIZE THE USE OF AN INSTRUMENT THAT CAN DIRECTLY READ AND DIGITIZE BETA EMISSIONS FROM (32)P ISOTOPECONTAINED ON HYBRIDIZED FILTERS TO INCREASE THE EFFICIENCY OF LIBRARY SCREENING. TWO METHODS WILL BE COMPARED THAT RESULT IN RESTRICTION SITE MAP DATA FOR COSMID CLONES ALLOWING OVERLAPS TO BE CONSTRUCTED FROM INDEPENDENTLY ISOLATED COSMIDS. STANDARD PROTOCOLS WILL BE ESTABLISHED FOR GENERATING THIS DATA AND KEY AREAS FOR AUTOMATION OF DATA PROCESSING USING THE DIRECT (32)P DETECTOR WILL BE IDENTIFIED. A LARGE SCALE IMPLEMENTATION OF THESE METHODS DURING PHASE II WILL BEGIN THE CONSTRUCTION OF A COMPLETE PHYSICAL MAP OF CHROMOSOME 21. A MAP OF CHROMOSOME 21 WILL SIGNIFICANTLY AID IN THE UNDERSTANDING OF THE ROLE OF THIS CHROMOSOME AND SPECIFIC GENES IN THE ETIOLOGY AND PATHOGENESIS OF DOWN SYNDROME AND ALZHEIMER'S DISEASE.