APPLIED BIOSYSTEMS MODEL 7700 01 SEQUENCE DETECTOR

此应用程序请求Perkin-Elmer ABI Prism 7700-01序列 配备Primer Express和序列检测软件的检测器。一个 额外的GeneAmp PCR系统960和一台Macintosh PowerMac 9500计算机 还要求有打印机的人在约翰配备一个中央设施 霍普金斯哮喘和过敏中心将由来自 总部设在中心的3个独立部门。这些调查人员 将使用序列检测器进行1)实时、定量RT-PCR 一系列与DIN炎症性疾病有关的细胞因子 航空公司；2)取代放射性等位基因特异性寡核苷酸 3)为基因分析提供了一种新的方法 转基因小鼠外源/内源基因拷贝的测定。 ABI棱镜系统的线性动态范围至少覆盖5 数量级，从而极大地减少了对串口 样品稀释度。该系统使用寡核苷酸探针，既有 一种报告染料和猝灭剂染料，它专门在 正向和反向引物。该探针被5‘核酸酶切割 Taq聚合酶释放报告染料的活性并产生 序列特定的信号。荧光强度与 一些放大的链条。目前，任何地方都不存在这样的系统 约翰·霍普金斯学院。 为了最大限度地提高拟议设施的效率，所有收购后的数据 分析将使用单独的PowerMac 9500计算机系统执行 它还将用作数据存储/服务器系统。200-MHz 微处理器将允许快速数据分析。最后，还有一个 要求GeneAmp 9600热循环以匹配闭管分析 ABI 7700系统使用的格式和循环条件。因此，样本 具有已知的序列变异，例如突变或多态 目前通过等位基因特异的寡核苷酸杂交检测到，或者 在9600型上进行聚合酶链式反应和限制性内切酶消化 并转移到ABI 7700进行探头检测。这将是 最大限度地提高ABI型号7700上的可用运行时间 需要实时定量应用的调查人员。 这项强大的新技术的推出将极大地帮助 研究了一大批富有生产力和互动性的教员。

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.