SUB-MICROLITER, HIGH THROUGHPUT THERMAL CYCLER

降低DNA测序成本以满足人类基因组计划所需目标的尝试取得了有限的成功，部分原因是循环测序和PCR所需的专有酶的持续高昂成本。尽管已经尝试通过稀释储备酶混合物和通过减少反应体积来降低循环测序成本，但是这些解决方案-如在市售热循环仪上实施的-并没有导致足够的成本降低，并且可能导致测序失败率的增加。此外，商业循环仪与全自动化实验室不兼容，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仪处理高于3穆尔的反应体积，导致热循环成本占总鸟枪测序成本的40%以上。随着改进的毛细管测序仪器现在可用，这些反应体积代表了获得良好序列数据所需的实质性过量。实验表明，小至目前使用的反应体积的1/10的体积可以产生优异的序列质量。该申请要求资金用于开发具有集成反应装置的高通量热循环仪，其能够以600个样品/小时/仪器的速率处理亚微升体积。该设备接收DNA模板和酶酿造物的微量滴定板作为输入，并自动设置和循环反应，将产物分配到预先填充有乙醇的微量滴定板中。通过在一次性毛细管内抽吸、混合和循环反应，仪器无需分配或抽吸小体积，除了从/到散装试剂。因此，避免了与小体积移液相关的表面张力限制，并可靠地移液亚微升反应。由于样品在密封的毛细管中循环，并被牺牲水样品包围，即使在300 nl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流循环-这避免了循环所需的样品板保持器的需要，并且与传统的热电循环仪相比显著增加了热斜坡率。拟议仪器的小容量能力将把热循环的消耗品成本从目前的1.00 - 1.50美元/样品降低到大约0.20美元/样品。由于该仪器将是全自动的，只需要很少的设置时间，因此也将节省大量的劳动力。仅考虑热循环耗材成本，这一发展预计将导致鸟枪测序的总成本（耗材+劳动力）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.