NON-RADIOACTIVE SOUTHERN BLOTTING DETECTION SYSTEM

非放射性印迹检测系统

• 批准号: 6072460
• 负责人: RICHARD DENIS POWELL
• 金额: $ 11.61万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6072460
• 关键词: biotin; chemical conjugate; chemical synthesis; fluoresceins; gold; human tissue; nanotechnology; nucleic acid hybridization; nucleic acid probes; southern blotting; technology /technique development

Fast, convenient detection and visualization reagents will be developed for Southern blotting, based upon the 1.4 nm Nanogold gold cluster label combined with autometallographic enhancement. In immunodot blotting Nanogold with autometallographic enhancement has already exhibited detection sensitivities comparable with autoradiography and chemiluminescence, and has visualized 0.1 pg (10[-18] mol) of a target IgG; In in situ hybridization, Nanogold with silver acetate autometallography has been used to visualize as few as 1-2 copies of target viral DNA in paraffin-embedded cells and biopsy sections for light microscopy. Three approaches will be tested: (a) detection of a haptenated hybridization probe with a secondary Nanogold conjugate; (b) Catalytic deposition of Nanogold-labeled substrates by an enzyme-conjugated hybridization probe; and (c) Catalyzed Reporter Deposition (CARD) with Nanogold-streptavidin as the final step using a haptenated probe. Nanogold will optimized by synthetic modification of its coordinated ligands for minimal non-specific binding and highest possible sensitivity. A novel gold-based autometallographic system which produces higher sensitivities and lower backgrounds will be used, and specialized blocking reagents developed to prevent non-specific membrane binding of either component. A reagent package and protocols will be developed for Southern blot hybridization detection in a few hours without film exposure, darkroom conditions or radioactivity. PROPOSED COMMERCIAL APPLICATION: In 1998, nearly 564,800 Americans are expected to die of cancer, and the annual costs for cancer are estimated to be $107 billion. Southern blotting is a primary method for detection and characterization of cancer: these reagents will enable faster, simpler Southern blotting for cancer diagnosis and research without the hazard and expense of radioactive probes. Their high spatial resolution may also lead to applications in high-resolution or high-throughput automated systems.

基于1.4nm纳米金团簇标记结合自显影技术，将开发用于Southern印迹的快速、方便的检测和可视化试剂。在免疫斑点印迹中，具有自显影增强的纳米金已经表现出与放射自显影和化学发光相当的检测灵敏度，并且已经可视化了0.1 pg（10[-18] mol）的靶IgG;在原位杂交中，具有银乙酸自显影的纳米金已经用于可视化石蜡包埋的细胞和活检切片中的靶病毒DNA的少至1-2个拷贝，用于光学显微镜。将测试三种方法：（a）使用二级纳米金缀合物检测半抗原化杂交探针;（B）通过酶缀合的杂交探针催化沉积纳米金标记的底物;和（c）使用半抗原化探针，作为最后步骤，使用纳米金-链霉亲和素催化报告物沉积（CARD）。纳米金将通过合成修饰其配位体来优化，以实现最小的非特异性结合和最高的灵敏度。将使用一种新的基于金的自动照相系统，该系统产生更高的灵敏度和更低的背景，并开发专门的阻断试剂以防止任何一种成分的非特异性膜结合。将在几个小时内开发出用于Southern印迹杂交检测的试剂包和方案，无需胶片曝光、暗室条件或放射性。拟定商业应用：在1998年，将近564，800名美国人预计将死于癌症，每年用于癌症的费用估计为1070亿美元。Southern印迹是检测和表征癌症的主要方法：这些试剂将使癌症诊断和研究的Southern印迹更快，更简单，而没有放射性探针的危险和费用。它们的高空间分辨率也可能导致在高分辨率或高通量自动化系统中的应用。