SYNTHETIC DNA AFFINITY MATRICES FOR HYBRIDIZATION ASSAYS

用于杂交测定的合成 DNA 亲和矩阵

• 批准号: 3497541
• 负责人: RICHARD H TULLIS
• 金额: $ 5万
• 依托单位: MOLECULAR BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 1985-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3497541
• 关键词: Alphaherpesvirinae; affinity chromatography; bacterial DNA; clinical biomedical equipment; clinical chemistry; communicable disease diagnosis; complementary DNA; diagnostic tests; microorganism classification; nucleic acid chemical synthesis; oligonucleotides; synthetic nucleic acid; virus DNA; virus classification

Our goal is to develop a rapid, simple DNA hybridization system for use in clinical samples. Current methods for performing hybridizations (eg. nitrocellulose dot blots) are difficult to apply to crude samples and require protracted sample preparation. One promising solution is the hybridization "sandwich" assay in which a first probe bound to a solid support (i.e. affinity matrix) selects the target DNA from the sample. A second probe then identifies the bound target. Synthetic DNA affinity supports, based on a new Teflon-polyacrylamide DNA synthesis resin, can be used as the affinity matrix. Synthetic DNA probes may also be used as the second probe. Such oligonucleotide sandwich supports are relatively inexpensive to produce. Phase I involves the synthesis and testing of oligonucleotide affinity support matrices complementary to Herpes simplex virus (HSV). Phase I will determine the sensitivity and specificity of oligomer probes for HSV-DNA bound to the affinity support matrix and their ability to differentiate HSV subtypes in the presence of heterologous DNA. Phase II will refine the use of DNA affinity supports using isotopic and non-isotopic probes to detect viruses and bacteria in clinical samples. Once sandwich assays are fully tested, we will produce clinical diagnostic tests for viruses and bacteria.

我们的目标是开发一种快速，简单的DNA杂交系统，用于 临床样本。 目前用于进行杂交的方法（例如， 硝酸纤维素斑点印迹法）难以应用于粗样品， 需要长时间的样品制备。 一个有希望的解决方案是 杂交"夹心"测定法，其中第一探针与固体结合 载体（即亲和基质）从样品中选择靶DNA。 一 然后第二探针鉴定结合的靶。 合成DNA亲和力 支持，基于一种新的聚四氟乙烯-聚丙烯酰胺DNA合成树脂，可以 用作亲和矩阵。 合成的DNA探针也可以用作探针。 第二个探头 这样的寡核苷酸夹心支持物相对 生产成本低。 第一阶段包括寡核苷酸亲和力的合成和测试 与单纯疱疹病毒（HSV）互补支持基质。 一期将 确定寡聚体探针对HSV-DNA的敏感性和特异性 结合到亲和支持基质和它们区分HSV的能力 在异源DNA的存在下的亚型。 第二阶段将使用同位素和同位素技术改进DNA亲和载体的使用， 非同位素探针检测临床样本中的病毒和细菌。 一旦夹心检测得到充分测试，我们将生产临床诊断试剂盒， 检测病毒和细菌