HOMOGENEOUS TIME-RESOLVED FLUORESCENCE DNA

THE AIM OF THIS STUDY IS TO DEVELOP A NONRADIOACTIVE, HOMOGENEOUS HYBRIDIZATION ASSAY FOR IDENTIFYING BACTERIAL AND VIRAL PATHOGENS IN CLINICAL SPECIMENS. HYBRIDIZATION WILL BE MONITORED USING A NOVEL TIME-RESOLVED FLUORESCENCE (TRF) INSTRUMENT TO DETECT FLUORESCENCE RESULTING FROM ENERGY TRANSFER BETWEEN DYES INTERCALATED INTO THE HYBRIDS (ENERGY DONORS) AND EU(3+) ATTACHED TO OLIGONUCLEOTIDE PROBES BY CHELATION (ENERGY ACCEPTORS). IN PHASE I, A MODEL SYSTEM CONSISTING OF POLY-DA TARGET SEQUENCES AND OLIGO-DT PROBES LABELED WITH EU(3+) WAS USED TO DEMONSTRATE THE FEASIBILITY OF THIS APPROACH. 9-AMINOACRIDINE WAS USED AS THE DONOR MOLECULE. OPTIMAL HYBRIDIZATION AND DETECTION CONDITIONS WERE DETERMINED, AND PRELIMINARY SENSITIVITY DETERMINATIONS WERE MADE. MORE DETAILED ANALYSES OF LABELING, HYBRIDIZATION, AND DETECTION PARAMETERS WILL BE PERFORMED IN PHASE II, WHERE THE ASSAY WILL BE APPLIED TO THE DETECTION OF BACTERIAL PATHOGENS IN SPECIMENS. THIS TRF-DNA ASSAY WILL BE FASTER, SIMPLER, AND AT LEAST AS SENSITIVE AS EXISTING RADIOACTIVE HYBRIDIZATION-DETECTION ASSAYS. IT REPRESENTS A SIGNIFICANT ADVANCE OVER THESE TECHNOLOGIES AND PROMISE TO HAVE CONSIDERABLE IMPACT ON THE DEVELOPMENT OF COMMERCIALLY RELEVANT DNA-BASED DIAGNOSTICS.