Novel methods to improve the acquisition of DNA sequence data, both from a cost reduction and throughput point of view, are still important to the field of genomics, in spite of the fact that a rough pass through the genome may be achieved by 2003. In this research the investigators are designing a modular nano-fluidic, high throughput DNA sequencing device that will allow the parallel processing of DNA samples in an automated fashion. The major functions that this device will have incorporated into it include PCR amplification of templates possessing a biotin tether in nano-liter reactors, purification of the PCR products in micro-chips using conductivity detection, immobilizing the biotinylated templates to a solid-phase nano-reactor, preparing Sanger, chain-terminated sequencing ladders in this nano-reactor, high speed fractionation of the ladders in micro-chips and fluorescence readout using near-IR detection. The unique aspect of the proposed approach is that the system will consume minimal amounts of costly reagents by preparing the samples on a volume-scale more commensurate with micro-column separation platforms. The nano-reactors are fabricated in conventional fused silica capillary tubes housed in rapid air thermal cyclers. Nanoliter volumes of reagents and samples are shuttled between devices using the electroosmotic flow generated by these capillaries. The investigators will also be fabricating micro-chips in plastics using LIGA processing, which will allow the fabrication of high aspect ratio microstructures with the added benefit that the devices can be injection molded. In this application, polymethylmethacrylate (PMMA) will be used as the substrate material, due to its ability to be readily injection molded and also, it can be functionalized through straightforward chemical procedures. The investigator's microchips will be used to purify PCR products using capillary electrochromatography and also, to fractionate sequencing ladders. The final phase of the research is the use of near-IR fluorescence readout with time-resolved discrimination for identifying bases in a single lane format. In addition, the proposed strategy will incorporate two-color chemistry. This, coupled with four lifetime discrimination, will allow the investigators to simultaneously monitor 8 spectroscopically distinct probes. Therefore, sequencing both ends of double-stranded templates to aid in mapping of YAC cosmids in order shotgun sequencing strategies can be carried out in a single read. The investigators will be preparing analogs of phthalocyanines (lambda(abs) = 670 nm) and naphthalocyanines (lambda (abs) = 780 nm) using heavy-atom modifications for controlling the lifetime. Fluorescence readout will be accomplished using simple diode lasers and avalanche diodes situated on a scanner head.
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