NON-RADIOACTIVE SOUTHERN BLOTTING DETECTION SYSTEM

点击翻译按钮获取中文摘要

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.