APPLIED BIOSYSTEMS MODEL 7700 01 SEQUENCE DETECTOR

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This application requests a Perkin-Elmer ABI Prism 7700-01 Sequence Detector equipped with Primer Express and Sequence Detection software. An additional GeneAmp PCR system 960 and a Macintosh PowerMac 9500 computer with printer are also requested to equip a central facility at the John Hopkins Asthma and Allergy Center to be used by funded investigators from 3 separate divisions that are based at the Center. These investigators will use the Sequence Detector for 1) Real-time, quantitative RT-PCR for a range of cytokines that are involve din inflammatory disease on the airways; 2) to replace radioactive-allele-specific oligonucleotide hybridization for genotype analysis; and 3) to provide a new method for determination of exogenous/endogenous gene copy in transgenic mice. The linear dynamic range of the ABI Prism system covers at least five orders of magnitude and thereby greatly reduces the need for serial dilutions of samples. The system uses an oligonucleotide probe with both a reporter and quencher dye attached, which anneals specifically between forward and reverse primers. The probe is cleaved by the 5' nuclease activity of Taq polymerase to release the reporter dye and generate a sequence-specific signal. Fluorescence intensity correlates directly with a number of amplified chains. Currently, no such system exists anywhere at the John Hopkins Institutions. To maximize efficiency of the proposed facility, all post-acquisition data analysis will be performed using a separate PowerMac 9500 computer system that will also serve as a data storage/server system. The 200-MHz microprocessor will permit rapid data analysis. Finally, an additional GeneAmp 9600 thermal cycle is requested to match the closed-tube assays format and cycle conditions used by the ABI 7700 system. Thus, samples with known sequence variations, such as mutations or polymorphisms currently detected by allele-specific oligonucleotide hybridization, or PCR followed by restriction enzyme digestion will be run on the model 9600 cycler and transferred to the ABI 7700 for probe detection. This will maximize available run-time on the ABI model 7700 for the multiple investigators who require real-time quantitative applications. The availability of this powerful new technology will greatly aid the research of a large group of productive and interactive faculty.