The goal of this project is to develop and optimize a direct, nonradiometric DNA hybridization detection system based upon the novel formation of sequence-specific, chemically synthesized oligonucleotide probes covalently linked to alkaline phosphatase. The long term objective is to deliver to the clinical market a rapid, sensitive and automated system for the determination of specific bacteria, virus and genetic abnormalities, whose identification by current immunoassay or culture technology is difficult or laborious. Specifically, the production of current and new oligomer-enzyme conjugates will be optimized. Conditions for maintaining stability and maximum enzyme activity under storage, hybridization and assay conditions will be identified. The use of fluorogenic substrates for increased sensitivity and automation compatability will be explored. Hybridization systems using filter membranes, bead solid supports or in solution will be developed using conjugate detection. Trials will be conducted with the conjugates for detecting viral and bacterial target DNA in clinical samples with the goal of marketing specific detection kits for various pathogens.
