ABI PRISM 7700 FOR TAQMAN PCR

西南生物医学研究基金会(SFBR)病毒学和免疫学系正在进行的几个由NIH支持的研究项目将得到极大的加强，因为可以使用ABI PRISM 7700序列检测器来执行Taqman、定量、实时PCR。TaqMan聚合酶链式反应是一种依赖于两个基本属性的定量聚合酶链式反应方法。在PCR扩增过程中使用5‘端核酸酶聚合酶链式反应，并实时监测分析过程中产生的荧光信号。5‘核酸酶分析类似于传统的扩增，增加了一个探针，对产生的PCR产物进行退火。该探针含有两个染料分子，即报告分子和猝灭剂。Taq的5‘核酸酶活性将使报告染料从退火的探针中释放出来，从而产生荧光信号。该信号在每个周期通过光纤连接到热循环仪上的孔(全部集成在ABI PRISM 7700中)进行测量。在每个PCR周期中的实时测量产生了5-6个对数范围的输入RNA或DNA分子的定量。这项技术适用于任何核酸的测量。在病毒学和免疫学系内，该仪器的主要用途将是确定实验感染动物、人类样本和组织培养系统中的病毒载量。第二个应用将是监测细胞因子和淋巴细胞亚群标志物的mRNA水平。该部门的研究包括人类及类似免疫缺陷病毒、猴T细胞白血病病毒I、人乙肝、丙型、丁型和戊型肝炎病毒、灵长类肝炎病毒、猴子疱疹B病毒、出血性竞技场病毒和感染利什曼原虫的病毒。在动物模型中评估这些病毒的致病机制、疫苗的开发和抗病毒药物的分析都依赖于病毒水平的测量。TaqMan聚合酶链式反应是目前定量聚合酶链式反应的首选方法。这将大大减少技术时间，同时提高数据质量。NIH赞助的这些研究人员的研究项目将因获得这种设备而大大增强。

Several NIH-supported research programs being conducted in the Department of Virology and Immunology at the Southwest Foundation for Biomedical Research (SFBR) would be greatly enhanced by access to an ABI PRISM 7700 sequence detector for the performance of Taqman, quantitative, real-time PCR. Taqman PCR is a method of quantitative PCR that relies on two essential attributes. The 5' nuclease PCR assay is used during PCR amplification and the fluorescence signal generated during the assay is monitored in real time. The 5' nuclease assay is similar to conventional amplification with the addition of a probe that anneals to the generated PCR product. The probe contains two dye molecules, the reporter and the quencher. The 5' nuclease activity of Taq will liberate the reporter dye from an annealed probe resulting in the generation of a fluorescent signal. This signal is measured in each cycle by fiber optics to the wells on the thermocycler (all integrated in the ABI PRISM 7700). Real time measurement in each cycle of PCR yields a quantitation over a 5-6 log range of input RNA or DNA molecules. This technology is applicable to the measurement of any nucleic acid. Within the Department of Virology and Immunology, the primary use of the instrument will be for the determination of viral load in experimentally infected animals, in human samples, and it tissue culture systems. A secondary application will be to monitor mRNA levels of cytokines and lymphocyte subset markers. The research of the department includes studies on human and similar immunodeficiency virus, simian T cell leukemia virus I, human hepatitis B, C, D and E viruses, a primate hepatitis virus, monkey herpes B virus, hemorrhagic arena- viruses, and a virus infecting Leishmania. Evaluation of the pathogenesis of these viruses in animal models, the development of vaccines and analysis of antivirals are all dependent upon the measurement of viral levels. TaqMan PCR is now the method of choice for quantitative PCR. It will result in a substantial reduction in technical time, while increasing the quality of the data. The NIH sponsored research programs of these investigators would be greatly enhanced by access to this equipment.