In this proposal we will develop methods to substantially increase the utility of dye-labeled terminator sequencing technology, and reduce the cost, by developing fluorescent dyes and modified dideoxynucleotide chain terminators. The proposed combination of a versatile acceptor nucleotide and compatible label presents a universal labeling system with a range of applications. In addition, non-sequencing applications are also anticipated. We expect applications for this approach in single strand conformation polymorphism analysis and automated dideoxy fingerprinting both of which are gaining clinical acceptance for rapid mutation detection (for example in tumor suppressor gene p53 and the breast cancer susceptibility gene BRCA1). Furthermore, the proposed technology will allow the convenient evaluation of newly developed fluorescent dyes without the need to generate their triphosphate derivatives. PROPOSED COMMERCIAL APPLICATION: A family of products will be derived from the proposed work: (i) novel dideoxy terminators for DNA sequencing, (ii) novel fluorescent dyes for DNA sequencing, (iii) Research kits for mutation detection. GRANT=R44RR12449 A Small Business Innovation Research Phase II program is proposed to complete the development and testing of an improved optical oxygen sensor for dissolved oxygen monitoring in bioreactors. The improved sensor, based on measurements of fluorescence lifetime quenching, will facilitate reliable, long-term control of oxygen levels in the media of culture and fermentation type reactors currently being used in the production of drugs and therapeutic proteins. The highly sensitivity indicator chemistry could also expand interest in newer oxygen sensitive culture systems which require oxygen concentration levels that are too low for reliable measurement using traditional oxygen electrodes. The specific aim of the Phase II effort is the fabrication and characterization of commercial prototype systems with the goal of confirming measurement resolution and stability adequate for use in bioreactor monitoring. Prototype sensors, constructed using a 12 man diameter design and all solid-state electro- optic components, will be evaluated for oxygen and temperature sensitivity using thermostatically controlled solutions equilibrated with certified gas mixtures of oxygen and nitrogen. Calibration routines and temperature correction algorithms will be developed and a series of beta tests conducted on the complete prototype systems in the operational bioreactors of industrial and university research groups. PROPOSED COMMERCIAL APPLICATION: Success in the applications of modern biotechnological techniques is evidenced by the variety of products, ranging from specialty chemicals, polymers and food, to therapeutic drugs produced by using recombinant DNA methodologies in various cell culture systems. There is a continuing need to develop and implement sensing systems to effectively control and optimize these biological processes. There also exists a need for water quality monitoring in hydroelectric and wastewater treatment facilities.
