HIGH-SPEED PCR USING A PRESSURIZED GAS THERMOCYCLER

描述（申请人摘要）：在I期研究中，使用加压气体 利用热循环仪成功地扩增了基因片段 从人β-珠蛋白（536 b. p.），人血小板抗原（364 bp）， 噬菌体λ（333 bp），和细菌病原体，大肠杆菌0157：H7（91 b. p.和85 b. p.扩增子）。在我们最快的实验中，一个短的85 bp。 在78秒内通过30个PCR循环扩增扩增子。 该装置的速度和热控制受到其简单的3阀的限制 配置（热气阀、冷气阀、节流阀）、流量 空调、气体冷却不足、软件和缺乏在线DNA 探测光学 在第二阶段研究中，改进的高性能4阀和5阀热循环仪 将配备在线荧光检测光学系统。简称（小于 100 b.p.）扩增子，将进行30个扩增/检测循环 在不到60秒的时间内完成更长的扩增子（100至1000个） 500 b.p.）将在1到4分钟内用压缩空气放大。 加压气体热循环仪将使基于DNA的遗传性和 传染性疾病在一个前所未有的时间尺度。 拟定商业应用： 该方法比最快的商用热空气快十倍 PCR热循环仪;比常用的热块热循环仪快100倍。 第二阶段研究将开发高通量筛选的使能技术 能力，可应用于诊断，药物发现和功能基因组学 与遗传性和传染性疾病有关。 有非常大的和不断增长的市场， 用于医学/兽医诊断、农业综合企业和生物医学研究的高速PCR。

DESCRIPTION (Applicant's abstract): In Phase I Research, a pressurized gas thermocycler was fabricated and successfully used to amplify gene fragments from human beta-globin (536 b.p.), Human Platelet Antigen (364 b.p.), bacteriophage lambda (333 b.p.), and a bacterial pathogen, E.coli 0157:H7 (91 b.p. and 85 b.p. amplicons). In our fastest experiments, a short 85 b.p. amplicon was amplified through 30 PCR cycles in 78 seconds. The speed and thermal control of this device were limited by its simple 3-valve configuration (hot gas valve, cold gas valve, throttle valve), flow conditioner, inadequate gas cooling, software, and lack of on-line DNA detection optics. In Phase II Research, improved high performance 4- and 5-valve thermocyclers will be fitted with on-line fluorescence detection optics. For short (less than 100 b.p.) amplicons, 30 cycles of amplification/detection will be carried out in less than 60 seconds using pressurized Helium gas. Longer amplicons (100 to 500 b.p.) will be amplified in 1 to 4 minutes using pressurized air. Pressurized gas thermocyclers will enable DNA-based diagnosis of heritable and infectious diseases on an unprecedented time scale. PROPOSED COMMERCIAL APPLICATION: The proposed method is ten times faster than the fastest commercially available hot-air PCR thermocyclers; and 100 times faster than commonly used heat block thermocyclers. Phase II Research will develop enabling technology for high-throughput-screening capabilities, which can be applied to diagnostics, drug discovery, and functional genomics related to heritable and infectious diseases. There is very large and growing market for high-speed PCR for medical/veterinary diagnostics, agribusiness, and biomedical research.