SUB-MICROLITER, HIGH THROUGHPUT THERMAL CYCLER

降低DNA测序成本以满足人类基因组计划所要求的目标的尝试取得了有限的成功，部分原因是循环测序和PCR所需的专有酶的成本仍然很高。虽然已经尝试通过稀释原液酶混合物和减少反应体积来降低循环测序成本，但这些解决方案（在商用热循环上实施）并没有导致足够的成本降低，而且可能导致测序失败率的增加。此外，商业循环器与全自动实验室不兼容，因为它们不能很好地与用于反应设置和小体积移液的机器人集成。蒸发和液体转移组织限制了这些循环器处理的反应量超过3mol，导致热循环成本占散弹枪测序总成本的40%以上。随着改进的毛细管测序仪器现在可用，这些反应体积代表什么需要获得良好的序列数据大量过剩。实验表明，只需目前使用的反应体积的1/10，就能产生极好的序列质量。本申请申请资金用于开发具有集成反应装置的高通量热循环器，能够以600个样品/小时/仪器的速度处理亚微升体积。该设备接收DNA模板和酶制剂的微量滴度板作为输入，并自动设置和循环反应，将产物分配到预先填充乙醇的微量滴度板中。通过在一次性毛细管内的抽吸、混合和循环反应，该仪器不需要从散装试剂中抽吸或抽吸小体积试剂。因此，避免了与小体积移液相关的表面张力限制，并且可靠地移液了亚微升反应。由于样品在密封的毛细血管中循环，并被牺牲的水样包围，即使在300nl体积下，蒸发也可以忽略不计。毛细管直接使用恒温流体或气体流动进行循环，这避免了循环所需的样品板支架，并且与传统的热电循环器相比，显着增加了热斜坡率。该仪器的小体积性能将使热循环的耗材成本从目前的1.00- 1.50美元/样品降低到约0.20美元/样品。由于仪器将完全自动化，需要很少的设置时间，也将有大量的劳动力节省。仅考虑热循环耗材成本，这一发展预计将使鸟枪测序的总成本（耗材+人工）降低33%。

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.