SOLID STATE DNA SIZING

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定尺，主要基于凝胶电泳。本提案的长期目标是开发基于原子力显微镜（AFM）的极其敏感，快速和高通量的DNA分级方法。AFM是目前唯一实用的单分子技术，可以准确地确定几千个碱基或更小的dna的长度。它也是唯一一种不需要增强剂就能检测单个DNA分子或与DNA结合的单个蛋白质的信噪比技术。与基于凝胶的方法相比，这可以减少几个数量级的样本量。此外，固定的DNA样品可以在不到2分钟的时间内用现有的仪器测量大小。因此，AFM提供了几个潜在的优势比目前和竞争的技术。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分级，并实现自动AFM对吸附在表面上的目标DNA的成像阵列的使用。需要施胶的DNA样本将被固定在一个坚固的支架上，并通过原子力显微镜成像。自动成像将利用高精度的样品平台，可以在14英寸的距离内将AFM尖端定位在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尺寸直方图。为了进一步开发AFM的功能，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修饰DNA片段的方法，这将在一定程度上确定DNA片段的身份。传统的限制性内切作图揭示了限制性内切位点的序列依赖性分布，是DNA修饰的一种间接形式。通过AFM直接可视化停滞的限制性内切酶的能力可以用来产生直接对应于限制性内切图的装饰模式，其他装饰策略将提供独特的编码模式。将开发图案识别软件来确定装饰图案，还将开发比较图案的软件。DNA修饰和固态分级的结合将用于订购来自bac或pac的质粒。这样的有序集可以减少冗余测序的数量，并有利于填补霰弹枪测序过程中产生的空白。

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.