Electrophoresis in capillary tubes is an important recent development. The potential for substantially increased speed and extended size range in sequencing runs have been demonstrated recently. A major problem is that of detection of the separated components. Another problem is to maintain the excellent performance over many runs in a rugged system. We are developing novel separation, detection, and imaging techniques for real-time monitoring in capillary electrophoresis. Emphasis will be on schemes that allow multiplexing and methods that do not require specialized tags or specialized biochemistry. These techniques will be used to substantially increase the speed, throughput, reliability, and sensitivity in DNA sequencing applications. We estimate that it should be possible to eventually achieve a raw sequencing rate of 40 million bases per day in one instrument based on the standard Sanger protocol. Our research group possesses the unique expertise for combining bioanalytical separations with sensitive optical spectroscopies, and is therefore particularly well suited for performing the proposed work. Four distinct but inter-related goals can be identified. 1. Optimization of excitation and emission optics for highly multiplexed DNA sequencing based on parallel electrophoretic runs in capillary gels. Innovative laser coupling schemes and detection by solid-state imaging devices will be evaluated to facilitate the eventual development of rugged, large scale sequencing instrumentation. 2. Acceleration of electrophoresis in capillary gels by gel-matrix programming, voltage programming, pulsed-field programming, and temperature programming. Insights into the surface chemistry of the capillaries and entanglement mechanisms in mixed-gel matrices will lead to improved reproducibility and performance of DNA separations in high- volume applications. 3. New data treatment and base-calling schemes based on standard DNA labels that will allow increased light throughput to the detector and improved accuracy. Emphasis will be put on-line noise reduction and peak recognition software to reduce data work-up and to increase data throughput rate. 4. Interface to mature front-end automation strategies so that the raw sequencing rate can be utilized immediately. This includes designing the proper loading format to the capillary array and modifying commercial laboratory workstations (with respect to time and space requirements) accordingly, to facilitate future development of a fully integrated strategy.
