Attempts to decrease DNA sequencing costs to meet the goals required by the Human Genome Project have had limited success, partially as a result of the continued thigh cost of proprietary enzymes required for cycle sequencing and PCR. Though attempts have been made to reduce cycle sequencing costs by diluting stock enzyme mixes and by reducing reaction volumes, these solutions-as implemented on commercially available thermal cyclers-do not lead to sufficient cost decrease and can lead to increases in sequencing failure rates. Furthermore, commercial cyclers are incompatible with fully automated laboratories as they do not integrate well with robots used for reaction set-up and small volume pipetting. Evaporation and liquid transfer tissues limit these cyclers to processing reaction volumes above 3 mul, leading to a thermal cycling cost that accounts for over 40% of the total shotgun sequencing cost. With improved capillary sequencing instruments now available, these reaction volume represent a substantial excess over what is required to achieve good sequence data. Experiments have shown that volumes as small as 1/10 of the presently use reaction volumes can yield excellent sequence quality. This application requests funds for the development of a high throughput thermal cycler with integrated reaction set-up capable of processing sub- microliter volumes at the rate of 600 samples/hour/instrument. This device receives microtiter plates of DNA template and enzyme brews as input and automatically sets up and cycles the reactions, dispensing the products into microtiter plates pre-filled with ethanol. By aspirating, mixing, and cycling the reactions within disposable capillary tubes, the instrument is never required to dispense or aspirate small volumes except from/to a bulk reagent. Consequently, the surface tension limitations associated with small volume pipetting are avoided and sub microliter reactions are pipetted reliably Since samples are cycled in sealed capillaries and surrounded by sacrificial water samples, evaporation is negligible even at 300 nl volumes. The capillaries are cycled directly using constant temperature fluid or gas flow-this obviates the need to cycle a need sample plate holder and dramatically increases thermal ramp rates compared to conventional thermoelectric cyclers. The small volume capability of the proposed instrument will reduce the consumables costs of thermal cycling to approximately $0.20/sample from the present cost of $1.00-$1.50/sample. Since the instrument will be fully automated and require very little set-up time, there will also be substantial labor savings. Considering only thermal cycling consumables costs, this development is expected to lead to a 33% reduction in the total cost (consumables + labor) of shotgun sequencing.
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