This research will focus on developing a highly multiplexed DNA sequencing device that will incorporate automated methods for amplifying the template by PCR in microtiter dishes; preparing nanoliter-sized volumes of Sanger extension products and purification of these oligonucleotides prior to electrophoresis; high speed separations of these sequencing ladders using micro-CE and PEO sieving buffers; NIR laser-induced fluorescence detection with the base-calling performed in a single lane, single fluor format (bases identified via fluorescence lifetime discrimination) and finally, integration and organization of the sequencing data into an electronic form for sequence finishing and entry into GenBank. The device will accept templates amplified via PCR in 96-well microtiter plates using a biotinylated, forward primer. The double-stranded PCR template will be immobilized onto the walls of fused-silica capillary tubes with volumes ranging from 100-200 nL. These tubes are modified with avidin, which serves as the anchor for immobilizing the dsDNAs. Once immobilized, the DNAs are purified and the Sanger products produced directly in this tube using solid-surface sequencing strategies. The Sanger products are denatured and injected directly into a multiplexed CE system for performing fast separations of the ladders with expected read lengths of 400-500 bases. The fluorescence is excited on-chip with optical fibers carrying the excitation light to an array of capillary channels and a second fiber for collecting the emission and subsequently processed in a serial fashion via mirrors directing the light onto the face of a single photon avalanche diode. The important attributes of our approach include; the requirement of nanoliter-sized volumes of expensive sequencing reagents and dye-labeled ddNTPs; automated processes for purifying the PCR products, preparing the sequencing ladders and injection on the CE array (minimization of sample handling); high speed separations; highly efficient base-calling in a single lane, single dye format; and easily integrated into various automated robotic platforms which perform sample cloning and isolation. We will also develop theoretical models to investigate the feasibility of performing free solution separations of end-labeled DNA and free-flowing polymeric buffers which do not require reloading of the separation matrix and column reconditioning after each sequencing run.
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