Rational Engineering of Nanopost Arrays for DNA Electrophoresis

DNA 电泳纳米柱阵列的合理工程

• 批准号: 7762906
• 负责人: Kevin D Dorfman
• 金额: $ 28.15万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7762906
• 关键词: Accounting; Area; Biological Models; Biology; Biomedical Research; Caliber; DNA; DNA Fingerprinting; Data; Device Designs; Devices; Electrophoresis; Engineering; Ensure; Gel; Generations; Genome; Genomics; Goals; Hour; Laboratories; Laws; Lead; Literature; Maps; Medicine; Methods; Microfluidics; Organism; Performance; Process; Protocols documentation; Province; Pulsed-Field Gel Electrophoresis; Radial; Reading; Relaxation; Research; Resolution; Sampling; Screening procedure; Shotgun Sequencing; Staging; System; Technology; Time; Translating; Translations; Video Microscopy; Work; base; cost; design; genome sequencing; improved; innovation; insight; migration; models and simulation; nanofluidic; nanoscale; pathogen; physical mapping; programs; public health relevance; research study; restriction enzyme; simulation; single molecule; tool; trend

DESCRIPTION (provided by applicant): Nanofluidic DNA separation media promise to reduce the time required to resolve long DNA (> 20 kbp) by size from days to minutes. Amongst these new media, the most promising and well-developed are "artificial gels" formed by arrays of cylindrical posts. However, the translation of nanofluidic devices from the proof-of-principle stage into routine use in biology and medicine has been hampered by an absence of engineering analyses that relate the device design to its performance. The long-term goal of this research program is to develop, from a systematic and quantitative basis, DNA electrophoresis devices whose geometries are specifically designed to separate a given DNA size-range. The objective of this particular application is to engineer nanopost arrays for sep- arating long DNA. To accomplish this goal, Brownian dynamics simulations will be used to design the devices, single-molecule videomicroscopy will be used to validate the simulations, and analytical separation experiments will be used to evaluate the device performance. Preliminary results, based on this approach, contradict the con- ventional wisdom that the posts need to be closely spaced or randomly distributed to ensure frequent collisions with the DNA. Rather, these preliminary data indicate that sparse ordered arrays have the potential to greatly increase the separation power of nanopost separation media. Based upon this insight, the research is plan is divided into four specific aims: Specific Aim 1: Demonstrate, via experiments, long DNA separations in a sparse hexagonal nanopost array; Specific Aim 2: Develop a quantitative simulation model and validate it with the latter experiments; Specific Aim 3: Use this simulation model and further experiments to evaluate a non-hexagonal array geometry; Specific Aim 4: Engineer the most promising geometry to size a relevant genomic sample. This research is significant because it will lead to improvements in the analysis of genomes. Although second- generation sequencers may ultimately prove to be the most suitable method for re-sequencing projects, DNA fingerprinting and physical mapping will remain important for the assembly of unknown genomes and low- cost screening applications. The devices produced by this research will impact genome analysis by providing optimized systems for sizing DNA in the range relevant for restriction digests of BAC clones. This work is innovative because it counters the current trend in nanofluidic devices towards dense arrays for DNA separations. The research takes advantage of a synergistic combination of focused experimentation and systematic simulation studies, a design approach which has not been employed thus far. Taken as a whole, this research program will impact the larger field of biomedical microfluidics and nanofluidics by providing a systematic engineering framework for translating DNA electrophoresis devices from the proof-of-principle stage to optimized devices. PUBLIC HEALTH RELEVANCE: The proposed work will lead to improved nanoscale systems for rapid and high-resolution separations of long DNA. These devices will accelerate a number of key genomics applications, such as DNA fingerprinting of infec- tious organisms and genome assembly.

描述（由申请人提供）：纳米流体DNA分离介质有望将解析长DNA（> 20 kbp）所需的时间从数天减少到数分钟。在这些新介质中，最有前途和发展良好的是由圆柱形柱阵列形成的“人工凝胶”。然而，纳米流体装置从原理验证阶段到生物学和医学中的常规使用的翻译受到了缺乏将装置设计与其性能相关联的工程分析的阻碍。该研究计划的长期目标是从系统和定量的基础上开发DNA电泳设备，其几何形状专门设计用于分离给定的DNA尺寸范围。该特定应用的目的是设计用于分离长DNA的纳米柱阵列。为了实现这一目标，布朗动力学模拟将用于设计设备，单分子视频显微镜将用于验证模拟，分析分离实验将用于评估设备性能。基于这种方法的初步结果与传统观点相矛盾，传统观点认为，柱子需要紧密间隔或随机分布，以确保与DNA频繁碰撞。相反，这些初步数据表明，稀疏有序阵列有可能大大增加纳米柱分离介质的分离能力。基于这一认识，该研究计划分为四个具体目标：具体目标1：通过实验证明稀疏六边形纳米柱阵列中的长DNA分离;具体目标2：开发定量模拟模型并通过后面的实验验证它;具体目标3：使用该模拟模型和进一步的实验来评估非六边形阵列几何形状;具体目标4：设计最有希望的几何形状来确定相关基因组样本的大小。这项研究意义重大，因为它将导致基因组分析的改进。尽管第二代测序仪可能最终被证明是重新测序项目的最合适方法，但DNA指纹图谱和物理作图对于未知基因组的组装和低成本筛选应用仍将是重要的。这项研究产生的设备将通过提供优化的系统来影响基因组分析，该系统用于在与BAC克隆的限制性内切酶相关的范围内对DNA进行大小测定。这项工作是创新的，因为它反对目前的趋势，在纳米流体装置对密集阵列的DNA分离。这项研究利用了集中实验和系统模拟研究的协同结合，这是一种迄今为止尚未采用的设计方法。总的来说，这项研究计划将通过提供一个系统的工程框架，将DNA电泳设备从原理验证阶段转换为优化设备，从而影响生物医学微流体和纳米流体的更大领域。 公共卫生相关性：拟议的工作将导致改进的纳米系统，用于快速和高分辨率的长DNA分离。这些设备将加速一些关键的基因组学应用，如感染性生物的DNA指纹和基因组组装。