ENZYME-DNA CONJUGATES FOR DIRECT DETECTION OF DNA HYBRIDS

DNA HYBRIDIZATION PROBES ARE USEFUL FOR DETECTING SPECIFIC BACTERIA, VIRUSES AND GENETIC ABNORMALITIES. IN ORDER TO FULLY UTILIZE THE POTENTIAL OF SUCH PROBES, A RAPID, SENSITIVE, NONRADIOACTIVE DETECTION METHOD IS NEEDED. RADIOAUTOGRAPHY CAN VISUALIZE 15 PICOGRAMS OF TARGET DNA IN OVERNIGHT EXPOSURES. ENZYME CONJUGATES WITH A HIGH TURNOVER NUMBER CAN GIVE EQUIVALENT SENSITIVITY IN A FEW HOURS (RENZ AND KURZ, NUCL. ACIDS RES. 12:3435, 1984). THE GOAL OF THIS RESEARCH IS TO DEVELOP A RAPID NONRADIOACTIVE DETECTION METHOD FOR DNA HYBRIDS. IN PHASE I WE WILL INVESTIGATE THE USE OF A DIRECT HYBRID DETECTION SYSTEM BASED ON ENZYMES COVALENTLY COUPLED TO A SYNTHETIC OLIGONUCLEOTIDE HYBRIDIZATION PROBE. THESE ENZYME-DNA CONJUGATES WILL BE TESTED IN DIRECT HYBRIDIZATION FORMATS WHICH SHOULD ELIMINATE THE NEED FOR THE TIME-CONSUMING AND INEFFICIENT SECONDARY (""INDIRECT'') TREATMENTS CURRENTLY USED. THE ENZYME CONJUGATE SHOULD BE DETECTABLE COLORIMETRICALLY USING VARIOUS STANDARD DYE SYSTEMS. IN PHASE II WE WILL OPTIMIZE THE SYNTHESIS OF THE CONJUGATE. WE WILL ALSO CONDUCT TRIALS OF THE COMPLEX FOR DETECTING VIRAL AND BACTERIAL DNA TARGETS IN CLINICAL SAMPLES WITH THE GOAL OF MARKETING SPECIFIC DETECTION KITS FOR VARIOUS PATHOGENS.