MODIFIED SITE-SPECIFIC ENDONUCLEASE FOR GENOME RESEARCH

WITH THE RAPID GROWTH OF HUMAN GENOME RESEARCH, THERE IS A GROWING NEED FOR ADVANCED MAPPING AND SEQUENCING METHODS. IN CURRENT METHODOLOGY, THERE IS NO SINGLE STEP PROCESS FOR GENERATING SPECIFIC DNA FRAGMENTS IN THE MEGABASE SIZE RANGE. PROMEGA WILL UNDERTAKE RANDOM MUTAGENESIS COUPLED WITH A POWERFUL, NOVEL GENETIC SELECTION TO ISOLATE FORMS OF AN INTRON-ENCODED SITE-SPECIFIC ENDONUCLEASE--I-PPO--WITH POTENTIAL FOR USE IN GENOME ANALYSIS. THESE GOAL OF THESE STUDIES WOULD BE TO DEVELOP 1) A FAMILY OF ENZYMES WITH A RANGE OF CUTTING FREQUENCIES IN THE 2-1--M EGABASE SIZE RANGE, 2) ADVANCED METHODS FOR ANALYZING NOVEL DNA-PROTEIN INTERACTION, AND 3) ADVANCED GENETIC SELECTION METHODOLOGY USEFUL FOR DETAILED ANALYSIS OF THE DETERMINANTS FOR RECOGNITION AND CATALYSIS BY OTHER SITE-SPECIFIC ENDONUCLEASES. TO DEMONSTRATE THE FEASIBILITY OF ACHIEVING THESE RESULTS, PHASE I RESEARCH WILL FOCUS ON TWO SPECIFIC AIMS: 1) SYSTEMATICALLY EXAMINE THE TOLERATION FOR BASE SUBSTITUTIONS WITHIN THE CANONICAL 15 BASE PAIR I-PPO RECOGNITION SEQUENCE. 2) DEMONSTRATE THE FEASIBILITY OF A GENETIC SELECTION FOR MUTANT ENZYMES WITH ALTERED SPECIFICITY. SUBSEQUENT TO PHASE I AND PHASE II STUDIES, PROMEGA WOULD COMMERCIALIZE THE RARE-CUTTING ENZYMES, AND SEEK STRATEGIC ALLIANCES TO INCORPORATE THE ENZYMES INTO AUTOMATED DNA MAPPING AND ANLYSIS APPLICATIONS.