Dynamics of DNA Barcoding in Nanochannels

纳米通道中 DNA 条形码的动力学

• 批准号: 8651508
• 负责人: Kevin D Dorfman
• 金额: $ 35.09万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-11 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8651508
• 关键词: Address; Adopted; Attention; Behavior; Chromosome inversion; Collaborations; Communities; Complement; Cost Analysis; Coupled; Coupling; DNA; DNA Sequence; DNA Sequence Rearrangement; Data; Detection; Development; Device Designs; Devices; Engineering; Equilibrium; Fluorescence; Fluorescence Microscopy; Generic Drugs; Genome; Genome Mappings; Genomics; Goals; High-Throughput Nucleotide Sequencing; Human Genome; Immobilization; Individual; Industry; Label; Laws; Lead; Length; Location; Malignant Neoplasms; Measurement; Measures; Methods; Mission; Modeling; Monte Carlo Method; National Human Genome Research Institute; Population; Protocols documentation; Public Health; Reading; Research; Resolution; Role; Sampling; Silicon Dioxide; Simulate; Single Nucleotide Polymorphism; Speed; Stretching; Surface; Techniques; Technology; Testing; Time; Variant; Video Microscopy; Width; Work; base; cost; data acquisition; genome sequencing; improved; innovation; nanochannel; nanofabrication; new technology; next generation; next generation sequencing; novel; pathogen; predictive modeling; prevent; prototype; public health relevance; research study; simulation; single molecule; theories; tool

DESCRIPTION (provided by applicant): Many important variations in the genomes between different individuals in a population are in the kilobase pair (kbp) to megabase pair range. As they often feature numerous repeat sequences and inversions, these variations are difficult to analyze using next generation DNA sequencing, and their length prevents using hybridization microarrays. DNA barcoding, where genomic information is "read" from single molecules of stretched DNA, is emerging as a key tool for high-throughput detection of such large-scale rearrangements. Nanochannels present an especially attractive approach to reading DNA barcodes; when the labeled DNA is injected into a nanochannel, it stretches out and fluctuates about its equilibrium extension. Nanochannels reduce the error in the measurement of the genomic distance between barcodes by sampling the statistically independent configurations resulting from these fluctuations. While there are theories for the extension and dynamics of DNA in very small (< 20 nm) and relatively large (> 500 nm) nanochannels, these theories are of little use for device engineering ! the small channels are difficult to fabricate and operate, whereas the larger channels do not provide enough stretching to resolve the barcodes. As a result, most devices operate between these two limits. The absence of any fundamental understanding of the behavior of DNA in channels from 100-500 nm in width is hindering the engineering and optimization of nanochannels used for DNA barcoding and proposed uses of nanochannel arrays as a platform for next generation sequencing. Specific Aim 1 will build upon substantial preliminary data to produce an experimentally validated model of DNA in confinement. Accomplishing this goal requires a tight integration of Monte Carlo simulations, Brownian dynamics simulations with fluctuating hydrodynamic interactions, nanofabrication and fluorescence videomicroscopy. In Specific Aim 2, this model will be used in conjunction with experiments on barcoded DNA to (i) optimize the resolution of the state-of-the-art protocol and (ii) test new, model-based protocols that should offer substantial advantages in cost and analysis time. This research is significant because it will greatly advance the understanding of confined DNA, in particular the dynamics of the chain. By resolving the open scientific questions, this research will advance DNA nanochannel array technologies for genomics. This technology focus is enhanced by a collaboration with BioNano Genomics. Accomplishing the specific aims requires an innovative synergistic coupling between different simulation methods and experimental techniques and partnership with industry. This work will impact the transition of nascent nanochannel technology to end-users in the genomics community through a rational engineering framework for optimizing barcode reading protocols. It is expected that the novel model-based measuring protocol produced from this research will lead to hundred-fold improvements in the analysis time while reducing the cost of the nanochannels, associated hardware and consumables.

描述(由申请人提供)：群体中不同个体之间基因组的许多重要变异在千碱基对(KBP)到兆碱基对的范围内。由于它们经常以大量重复序列和倒置为特征，这些变异很难使用下一代DNA测序进行分析，而且它们的长度阻碍了使用杂交微阵列。DNA条形码是从拉伸的DNA单分子中读取基因组信息的方式，它正在成为高通量检测这种大规模重排的关键工具。纳米通道提供了一种特别吸引人的读取DNA条形码的方法；当标记的DNA被注入纳米通道时，它会伸展并围绕其平衡延伸波动。纳米通道通过对由这些波动产生的统计上独立的构型进行采样来减少测量条形码之间的基因组距离的误差。虽然有关于DNA在非常小(&lt；20 nm)和相对较大(&gt；500 nm)纳米通道中的延伸和动力学的理论，但这些理论在设备工程中几乎没有用处！较小的通道难以制造和操作，而较大的通道不能提供足够的拉伸来解析条形码。因此，大多数设备都在这两个限制之间运行。对DNA在100-500 nm宽度的通道中的行为缺乏任何基本的了解，这阻碍了用于DNA条形码的纳米通道的工程设计和优化，并建议使用纳米通道阵列作为下一代测序的平台。特定目标1将建立在大量初步数据的基础上，以产生一个经过实验验证的禁闭中的DNA模型。要实现这一目标，需要将蒙特卡罗模拟、布朗动力学模拟与波动流体动力学相互作用、纳米制造和荧光视频显微镜紧密结合在一起。在具体目标2中，该模型将与条形码DNA实验结合使用，以(I)优化最先进协议的分辨率和(Ii)测试新的、基于模型的协议，这些协议应在成本和分析时间方面提供实质性优势。这项研究意义重大，因为它将极大地促进对受限DNA的理解，特别是链的动力学。通过解决悬而未决的科学问题，这项研究将推动DNA纳米通道阵列技术在基因组学中的应用。通过与生物纳米基因组公司的合作，这一技术重点得到了加强。具体目标的实现需要不同的模拟方法和实验技术之间的创新协同耦合，并与产业界建立伙伴关系。这项工作将通过优化条形码读取协议的合理工程框架，影响新生的纳米通道技术向基因组学社区最终用户的过渡。预计这项研究产生的基于模型的新型测量协议将使分析时间提高数百倍，同时降低纳米通道、相关硬件和消耗品的成本。