SITE-SPECIFIC ENDONUCLEASES FOR HUMAN GENOME MAPPING

A MAJOR LIMITATION IN CURRENT LARGE SCALE GENOME MAPPING METHODOLOGY IS THAT FEW TOOLS EXIST FOR GENERATING SPECIFIC DNA FRAGMENTS IN THE MEGABASE SIZE RANGE. THIS PHASE I RESEARCH IS EXAMINING THE FEASIBILITY OF DEVELOPING A FAMILYOF INTRON-ENCODED SITE-SPECIFIC ENDONUCLEASES CAPABLE OF GENERATING DNA FRAGMENTS 2 TO 100 MEGABASES IN SIZE BY USINGTHE FOLLOWING STEPS: (1) DEVELOP METHODS FOR EXPRESSION OF I-PPO (AN ENDONUCLEUS FROM PHYSARUM POLYCEPHALUM) IN E. COLIAT LEVELS NECESSARY TO SUPPORT FURTHER STUDY AND EVENTUAL COMMERCIALIZATION OF THE ENZYME; (2) PURIFY THE INTRON-ENCODED ENDONUCLEASE I-PPO TO DETERMINE SUITABILITY AS A MOLECULAR BIOLOGY REAGENT ENZYME; (3) DEVELOP A RAPID, QUANTITATIVE ASSAY TO DETERMINE INITIAL VELOCITY OF I-PPO; (4) OPTIMIZE REACTION CONDITIONS, EXAMINE STABILITY, AND CHARACTERIZE OTHER PROPERTIES RELEVANT TO MEGABASE MAPPING METHODOLOGY; AND (5) ASSESS THE ENZYME'S ACTIVITY AND SPECIFICITY IN AN AGAROSE MATRIX AS WELL AS THE ABILITY TO CLEAVE COMPLEX GENOMIC DNA. PHASE II RESEARCH WILL INCLUDE STUDIES TO DEVELOP I-PPO FOR THE INTENDED APPLICATION AND WILL FOCUS ON CUTTING FREQUENCY AND SPECIFICITY, PARTIAL DIGESTION AND STAR ACTIVITY, PROTEIN MODIFICATION THROUGH CHEMICAL MODIFICATION AND LIMITED PROTEOLYSIS AS NEEDED, ANDINTERACTIONS OF THE ENZYME WITH THE TARGET SITE TO IDENTIFY SPECIFIC CHANGES IN THE PROTEIN THAT ALTER SPECIFICITY.