ONE MINUTE PCR FOR MEDICAL DIAGNOSTICS

聚合酶链式反应(PCR)是分子生物学中应用最广泛的技术之一。一般来说，大多数热循环仪依赖于具有较大热质量的可编程热块，因此，在PCR循环中的大部分时间都在变性、退火和伸长温度之间非生产性地度过。最近，建造了速度更快的热风热循环器，缩短了这些转换时间，允许在10到30分钟内完成30个循环的PCR。虽然这些系统的设计在原则上是优雅的，但并不是最优的。空气是一种相对较差的换热介质，在大气压下，单个热室/反应室的运行固有地很慢。利用计算机控制的电子阀将加压氦气输送到反应室，可以制造出速度更快的热循环器。在第一阶段研究中，将制造和测试加压氦气热循环器。性能将通过放大85 b.p来展示。人血小板抗原(HPA-4)扩增子和一个107b.P.E.Coli0157：H7 uidA扩增子。这些实验将在不到一分钟的时间内证明使用快速气相聚合酶链式反应对遗传性和传染性疾病进行高通量筛查(HTS)诊断的可行性。建议的商业应用：建议的方法比商业上最快的热风PCR热循环器快10倍，比常用的热块热循环器快100倍。第一阶段研究将开发高通量筛选(HTS)能力的使能技术，可应用于与遗传和传染病相关的诊断、药物发现和功能基因组学。用于医疗/兽医诊断和生物医学研究的高速聚合酶链式反应有着巨大且不断增长的市场。

The Polymerase Chain Reaction (PCR) is one of the most widely used techniques in molecular biology. In general, most thermocyclers rely upon programmable heat blocks which have a large thermal mass consequently, most of the time in a PCR cycle is spent non-productively in transition between denaturation, annealing, and elongation temperatures. Recently, much faster hot-air thermocyclers have been constructed which shorten these transition times, allowing 30 cycles of PCR in 10 to 30 minutes. While elegant in principle, the design of these systems is not optimal. Air is a relatively poor heat transfer medium, and the operation of a single heat/reaction chamber at atmospheric pressure is inherently slow. Much faster thermocyclers can be constructed using pressurized helium gas delivered to a reaction chamber by computer-controlled electronic valves. In Phase I Research, a pressurized helium gas thermocycler will be fabricated and tested. Performance will be demonstrated by amplification o f an 85 b.p. human platelet antigen (HPA-4) amplicon and a 107 b.p. E. coli 0157:H7 uidA amplicon. These experiments will demonstrate the feasibility of using rapid gas phase PCR for high-throughput-screening (HTS) diagnostics of heritable and infectious disease in less than one minute. PROPOSED COMMERCIAL APPLICATIONS: The proposed method is 10 times faster than the fastest commercially available hot-air PCR thermocyclers; and 100 times faster than commonly used heat block thermocyclers. Phase I Research will develop enabling technology for high-throughput-screening (HTS) capabilities, which can be applied to diagnostics, drug discovery, and functional genomics related to heritable and infectious diseases. There is large and growing market for high-speed PCR for medical/veterinary diagnostics and biomedical research.