This shared instrument grant application requests an ABI PRISM 7700 Sequence Detection System for use by six groups of investigators working in the disciplines of cell signaling, molecular biology, neurobiology, genetics, immunology and microbial pathogenesis for their studies of regulated gene expression and single nucleotide polymorphisms. The system integrates a polymerase chain reaction (PCR)-based thermal cycler with hardware instrumentation employing laser detection of fluorochromes and software. It will provide this group of investigators with a system for real-time quantitation of nucleic acid sequences with accuracy and reproducibility over a wide dynamic range and with high-throughput. By combining thermal cycling, fluorescence detection, and software in a single instrument, the system enables cycle-by-cycle detection of increases in nucleic acid product. In addition, data output is available immediately following sequence amplification without the need for additional purification steps or analysis by gel electrophoresis. Real time quantitative detection of specific nucleic acid sequences is based on the use of fluorescent probes incorporating reporter and quencher dyes on the same probe, with probe cleavage based on the 5' nuclease activity of Taq DNA polymerase. Probe cleavage separates the reporter dye from the quencher, thereby restoring fluorescence. The instrument's spectrograph and a charge-coupled device camera detect fluorescence emission. The system uses a 96-sample well platform, an argon laser to induce fluorescence, and a multiplexed array of optical fibers. The components include: (1) a 7700 sequence detector with built-in thermal cycler with 96-well positions; (2) an apple power Macintosh system with 16Mb RAM, 500-Mb hard disk drive, 120-MHz Power Macintosh, 1.4-Apple Super Drive floppy disk drive, CD ROM drive, 15"""""""" color monitor, and Hewlett Packard 1600C color deskjet printer. Sequence detection software used for instrument control, data collection, and automatic data analysis. The ABI PRISM 7700 System will be a crucial core resource for PHS-supported research on the Medical School campus of the Univ. of California at San Diego.
