Dynamics of DNA Barcoding in Nanochannels

描述（由申请人提供）：群体中不同个体之间的基因组中的许多重要变异都在核糖核酸酶对（kbp）至兆碱基对范围内。由于它们通常具有许多重复序列和倒位，这些变异难以使用下一代DNA测序进行分析，并且它们的长度阻止使用杂交微阵列。DNA条形码，其中基因组信息是从拉伸DNA的单分子中“读取”的，正在成为高通量检测这种大规模重排的关键工具。纳米通道是一种特别有吸引力的阅读DNA条形码的方法;当标记的DNA被注入纳米通道时，它会伸展并在其平衡延伸范围内波动。纳米通道通过对由这些波动产生的统计学上独立的构型进行采样来减少条形码之间的基因组距离测量中的误差。虽然存在DNA在非常小（< 20 nm）和相对大（> 500 nm）的纳米通道中的延伸和动力学理论，但这些理论对器件工程几乎没有用处！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，大多数设备在这两个限制之间运行。对宽度为100-500 nm的通道中DNA行为的任何基本理解的缺乏阻碍了用于DNA条形码化的纳米通道的工程化和优化以及纳米通道阵列作为下一代测序平台的拟议用途。具体目标1将建立在大量的初步数据，以产生一个实验验证的模型，DNA的限制。实现这一目标需要紧密集成蒙特卡洛模拟，布朗动力学模拟与波动的流体动力学相互作用，纳米纤维和荧光视频显微镜。在具体目标2中，该模型将与条形码DNA实验结合使用，以（i）优化最先进方案的分辨率，（ii）测试新的基于模型的方案，这些方案应在成本和分析时间方面具有实质性优势。这项研究意义重大，因为它将大大推进对受限DNA的理解，特别是链的动力学。通过解决开放的科学问题，这项研究将推进基因组学的DNA纳米通道阵列技术。这一技术重点通过与BioNano Genomics的合作得到加强。实现这些具体目标需要不同模拟方法和实验技术之间的创新协同耦合以及与工业界的伙伴关系。这项工作将通过优化条形码阅读协议的合理工程框架，影响新生纳米通道技术向基因组学界最终用户的过渡。预计从这项研究中产生的新的基于模型的测量协议将导致分析时间的百倍改进，同时降低纳米通道，相关硬件和耗材的成本。

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.