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.
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.
