The successful synthesis of a catalytic center nucleotide, incorporation of the nucleotide into a DNA probe and sensitive (5 attomol) detection of DNA targets in Phase I demonstrated the feasibility of developing a broad range of direct, sensitive nonradioactive nucleic acid detection assays for research and diagnostics. Concerns about the safety of individuals working repeatedly with radiolabeled nuclides, the increasing cost and difficulty of disposing of radioactive waste and the labor intensive aspects of current indirect nonradioactive detection methods confirms the need for direct detection systems such as the catalytic center system. In this Phase II proposal, we intend to extend the Phase I studies by further improving the sensitivity of the system and refining nucleic acid labeling, Southern and northern blotting and PCR product detection applications. These goals will be accomplished by synthesis and incorporation of catalytic centers with higher activity , use of chemiluminescent substrates with higher light yield and inclusion of enhancers to boost light signal. Catalytic center technology will antiquate radioactive detection methods and replace current nonradioactive detection methods which employ the use of secondary reagents such as antibody-enzyme conjugates. These benefits will provide scientists with the incentive to switch from current detection procedures to the catalytic centers system.
The potential commercial market is large considering that the use of 32P, 35S and 3H in U.S. research laboratories alone is conservatively estimated to be $60M annually. Additionally, radioactive disposal costs are estimated to range between $200M and $500M in the U.S. The availability of direct, sensitive nonradioactive detection methods will save many of these disposal dollars for the purchase of additional nonradioactive detection reagents.
