BIOLUMINESCENT ASSAY FOR QUANTIFYING PCR PRODUCTS

描述(改编自申请者摘要)：聚合酶链式反应的力量 反应在于它能够扩增极少量的DNA 生产足够的量以供测量。不幸的是， 放大通常会产生更大的误差 增加循环次数，以便在精确度之前损失精确度 测量是可能的。建议的解决方案是减少对 通过采用更灵敏的检测进行扩增。SeaLite Science拥有 开发了一种闪光型生物发光标签AquaLite。伟大的 AquaLite检测的灵敏度使其成为可能，在初步 研究，开发高精度、快速和廉价的定量 聚合酶链式反应和逆转录-聚合酶链式反应检测。这个项目将汇集 AquaLite的开发人员和具有多年经验的调查人员 聚合酶链式反应和逆转录-聚合酶链式反应，以完善这些分析，使它们 可以实现自动化，并以当代的简易性和精确度 免疫分析。具体的目标是首先开发一种能够 在4小时内定量测量特定DNA，其系数为 偏差小于15%。第二，建立了一种RT-PCR检测方法 人外周血细胞细胞因子信息的定量分析 针吸活检组织。该项目将通过一系列步骤进行，以 优化聚合酶链式反应，确定最佳DNA捕获方法 微米板，优化杂交条件，优化探针 生物发光反应的检测和效率优化。这个 第二个目标将通过评估提取RNA的方法来实现 从组织来看，检测聚合酶链式反应所需的RNA浓度标准化 产品、生物发光检测和鉴定的优化 理想的RT-PCR对照。在第二阶段，这些方法将用于开发 细胞因子和其他分析物的商业分析。

DESCRIPTION (Adapted from applicant's abstract): The power of the PCR reaction lies in its ability to amplify vanishingly small amounts of DNA to produce quantities sufficient for measurement. Unfortunately, amplification typically produces errors that become greater with increasing number of cycles so that accuracy is lost before accurate measurement is possible. The proposed solution is to reduce the need for amplification by employing more sensitive detection. SeaLite Sciences has developed a flash type bioluminescent tag, AquaLite. The great sensitivity of AquaLite detection has made it possible, in preliminary studies, to develop highly accurate, rapid and inexpensive quantitative PCR and RT-PCR assays. This project will marshal the expertise of the developers of AquaLite with investigators with many years experience in PCR and RT-PCR reactions to perfect these assays to the point that they can be automated and used with the ease and precision of contemporary immunoassays. The specific aims are first, to develop an assay that can quantitatively measure specific DNA in 4 hours with a coefficient of variations less than 15%. And second, to develop an RT-PCR assay for quantitative analysis of cytokine message from peripheral blood cells of needle biopsy tissue. The project will proceed via a series of steps to optimize PCR reaction, determine optimal means of capturing DNA on micrometer plates, optimize hybridization conditions, optimize probe detection and optimize efficiency of the bioluminescent reaction. The second aim will be approached by evaluating methods for extraction of RNA from tissue, standardization of RNA concentration necessary to detect PCR products, optimization of bioluminescent detection and identification of ideal RT-PCR controls. In Phase II, these methods will be used to develop commercial assays for cytokines and other analytes.