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

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（改编自研究者摘要）：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。基因组分析中最广泛使用的分析方法之一是DNA大小测定，主要基于凝胶电泳。本提案的广泛的长期目标是开发基于原子力显微镜（AFM）的极其灵敏、快速和高通量的DNA大小测定方法。原子力显微镜（AFM）是目前唯一能准确测定几个碱基或更小的DNA长度的实用单分子技术。它也是唯一一种具有信噪比的技术，可以检测单个DNA分子或与DNA结合的单个蛋白质，而无需造影剂。与基于凝胶的方法相比，这允许样品量减少几个数量级。此外，固定的DNA样品可以在不到2分钟的时间内与目前的仪器大小。因此，与现有技术和竞争技术相比，原子力显微镜具有几个潜在优势。研究人员建议开发一种简单的高密度样品沉积系统，用于基于AFM的DNA尺寸测量，并使用自动AFM对吸附在表面上的目标DNA阵列进行成像。将需要大小测定的DNA样品以密集阵列固定在固体支持物上并通过AFM成像。自动成像将利用高精度的样品台，可以将AFM针尖定位在14英寸距离内的1微米以内。模式识别软件将确定DNA长度的分布，并产生类似于凝胶密度扫描的大小直方图。为了进一步利用AFM的能力，研究人员将开发使用蛋白质、核酸或其他序列特异性试剂以序列特异性模式装饰DNA片段的方法，这将以一定程度的置信度指定DNA片段的身份。传统的限制性酶切图谱揭示了限制性酶切位点的序列依赖性分布，并且是DNA修饰的间接形式。通过AFM直接可视化停滞的限制性酶的能力可用于产生直接对应于限制性图谱的装饰图案，并且其他装饰策略将提供独特的编码图案。将开发模式识别软件以确定装饰模式，还将开发比较模式的软件。DNA修饰和固态大小测定的组合将用于排序源自BAC或PAC的质粒组。这样的有序集合将减少冗余测序的量，并促进鸟枪测序期间产生的空位的填充。