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大的热质量的可编程热块，因此，PCR循环中的大部分时间非生产性地花费在变性、退火和延伸温度之间的过渡中。最近，已经构建了更快的热空气热循环仪，其缩短了这些过渡时间，允许在10至30分钟内进行30个PCR循环。虽然原则上很优雅，但这些系统的设计并不是最佳的。空气是相对较差的传热介质，并且在大气压下的单个加热/反应室的操作固有地缓慢。使用由计算机控制的电子阀输送到反应室的加压氦气，可以构建更快的热循环仪。在第一阶段的研究中，将制造和测试一个加压氦气热循环仪。将通过扩增85 b. p.人血小板抗原（HPA-4）扩增子和107 b. p. E扩增子证明性能。coli 0157：H7 uidA扩增子。这些实验将证明在不到一分钟的时间内使用快速气相PCR进行遗传性和传染性疾病的高通量筛选（HTS）诊断的可行性。拟议的商业应用：所提出的方法比最快的市售热空气PCR热循环仪快10倍;比常用的热块热循环仪快100倍。第一阶段研究将开发高通量筛选（HTS）能力的使能技术，该技术可应用于与遗传性和传染性疾病相关的诊断、药物发现和功能基因组学。用于医学/兽医诊断和生物医学研究的高速PCR具有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.