Research Project
6404953
DESCRIPTION (provided by applicant): Currently available high throughput DNA sequencing instruments achieve base calling by wavelength resolved, fluorescence detection of dye labeled fragments using a CCD camera. Identification of labels using fluorescence lifetimes is also possible. Because cost effective, high speed, multichannel detectors are not available, adapting lifetime detection to high throughput sequencing would seem to require timesharing of a single element detection system between multiple electrophoresis channels. Southwest Sciences has developed a heterodyne fluorescence lifetime method that is readily scalable to multi-element detection. This approach uses simple, relatively low cost detection electronics and a diode laser excitation source and will perform competitively with current instrumentation at lower cost. In Phase I, we successfully demonstrated the necessary time resolution, sensitivity, and speed needed for DNA sequencing applications using a single element detector and aqueous dye solutions. The Phase II effort will focus on adapting this technique to capillary array sequencing. A single-capillary instrument with an optimized heterodyne detector will be used to investigate the base calling accuracy of this method in four-dye, single lane sequencing. Analysis methods will be developed to call bases from the lifetime data. Finally, a 16 element heterodyne detector, suitable for capillary arrays, will be constructed and evaluated. PROPOSED COMMERCIAL APPLICATION: Automated DNA sequencing has application to genetic testing for medical diagnostics, agricultural and pharmaceutical applications. Our heterodyne approach to DNA base calling will significantly lower the cost of automated DNA sequencing instrumentation.
