SOLID STATE DNA SIZING

固态 DNA 尺寸测定

• 批准号: 2803953
• 负责人: JAN H HOH
• 金额: $ 16.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2803953
• 关键词: DNA footprinting; atomic force microscopy; biomedical automation; computer assisted sequence analysis; method development; nanotechnology; nucleic acid sequence; nucleic acid structure; plasmids; DNA footprinting; atomic force microscopy; biomedical automation; computer assisted sequence analysis; method development; nanotechnology; nucleic acid sequence; nucleic acid structure; plasmids

DESCRIPTION (Adapted from the Investigator's Abstract): The nucleotide sequence of the human genome will provide health benefits ranging from new diagnostic tools to therapeutic reagents. One of the most widely used analytical methods in genomic analysis is DNA sizing, primarily based on gel electrophoresis. The broad long-term goal of the present proposal is to develop extremely sensitive, rapid and high throughput DNA sizing methods based on atomic force microscopy (AFM). The AFM is at present the only practical single molecule technology that can accurately determine the lengths of DNAs that are several kilobases or smaller. It is also the only technology with the signal-to-noise to detect single DNA molecules, or single proteins bound to DNA, without contrast enhancing agents. This allows for a reduction in sample size of several orders of magnitude, compared with gel based methods. In addition, an immobilized DNA sample can be sized in less than 2 minutes with current instrumentation. Thus the AFM offers several potential advantages over present and competing technologies. The investigators propose to develop a simple high-density sample deposition system for AFM-based DNA sizing, and implement the use of an automated AFM for imaging arrays of target DNAs adsorbed to a surface. DNA samples requiring sizing will be immobilized in dense arrays on a solid support and imaged by AFM. The automated imaging will utilize a high-precision sample stage that can position an AFM tip to within 1 micrometer over a 14-inch distance. Pattern recognition software will determine the distribution of DNA lengths, and produce a size histogram similar to densitometric scans of gels. To further exploit the capabilities of the AFM, the investigators will develop approaches to decorate DNA fragments in sequence-specific patterns using proteins, nucleic acids or other sequence-specific reagents, that will specify the identity of a DNA fragment with some level of confidence. Conventional restriction mapping reveals a sequence dependent distribution of restriction sites, and is an indirect form of DNA decoration. The ability to directly visualize stalled restriction enzymes by AFM can be used to produce decoration patterns that directly correspond to restriction maps, and other decoration strategies will provide unique coding patterns. Pattern recognition software will be developed to determine decoration patterns, and software that compares patterns will also be developed. The combination of DNA decoration and solid state sizing will be used to order sets of plasmids derived from BACs or PACs. Such ordered sets will reduce the amount of redundant sequencing, and facilitate the filling of gaps generated during shotgun sequencing.

描述（根据研究者的摘要改编）：人类基因组的核苷酸序列将提供从新的诊断工具到治疗试剂的健康益处。基因组分析中使用最广泛的分析方法之一是DNA尺寸，主要基于凝胶电泳。本提案的长期长期目标是基于原子力显微镜（AFM）开发极敏感，快速和高吞吐量DNA尺寸方法。 AFM目前是唯一可以准确确定几千酶或更小的DNA长度的实用单分子技术。它也是唯一具有信号到噪声的技术，可以检测单个DNA分子或与DNA结合的单蛋白，而无需对比度增强剂。与基于凝胶的方法相比，这可以减少几个数量级的样本量。另外，固定的DNA样品可以在不到2分钟的情况下使用当前的仪器进行尺寸。因此，AFM在当前和竞争技术方面具有多种潜在的优势。研究人员建议为基于AFM的DNA尺寸开发一个简单的高密度样品沉积系统，并实施使用自动化的AFM对目标DNA的成像阵列的使用。需要进行尺寸的DNA样品将固定在固体支撑的密集阵列中，并由AFM成像。自动成像将利用高精度样本阶段，该阶段可以将AFM尖端定位在14英寸距离内的1千米内。模式识别软件将确定DNA长度的分布，并产生类似于凝胶光密度扫描的尺寸直方图。为了进一步利用AFM的能力，研究人员将使用蛋白质，核酸或其他序列特异性试剂来开发以序列特异性模式来装饰DNA片段的方法，这些试剂将指定具有某种置信度的DNA片段的身份。常规限制映射揭示了限制位点的序列依赖性分布，并且是DNA装饰的一种间接形式。 AFM直接可视化停滞的限制酶的能力可用于产生直接与限制图相对应的装饰模式，而其他装饰策略将提供独特的编码模式。将开发模式识别软件来确定装饰模式，并且还将开发比较模式的软件。 DNA装饰和固态尺寸的组合将用于订购源自BAC或PAC的质粒集。这样的有序集将减少冗余测序的量，并促进弹枪测序期间产生的间隙的填充。