Dynamics of DNA Barcoding in Nanochannels

描述（由申请人提供）：种群中不同个体之间的许多重要基因组变异在千碱基对（kbp）到兆碱基对范围内。由于它们通常具有大量的重复序列和反转，这些变异很难用下一代DNA测序来分析，而且它们的长度也阻碍了杂交微阵列的使用。DNA条形码是一种从拉伸DNA的单分子中“读取”基因组信息的方法，它正在成为高通量检测此类大规模重排的关键工具。纳米通道提供了一种特别有吸引力的读取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。纳米通道通过采样由这些波动引起的统计独立构型，减少了测量条形码之间基因组距离的误差。虽然有关于DNA在非常小（< 20nm）和相对较大（bbb500nm）纳米通道中的延伸和动力学的理论，但这些理论对器件工程几乎没有用处！小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极限之间运行。缺乏对DNA在100- 500nm宽通道中的行为的基本理解，阻碍了用于DNA条形码的纳米通道的工程和优化，以及纳米通道阵列作为下一代测序平台的建议使用。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DNA禁闭模型。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起来。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，（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。通过解决这些尚未解决的科学问题，本研究将推动基因组学中DNA纳米通道阵列技术的发展。通过与BioNano Genomics的合作，这种技术重点得到了加强。实现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读取协议的合理工程框架，影响新兴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新的基于模型的测量协议将导致分析时间的数百倍改进，同时降低纳米通道、相关硬件和消耗品的成本。

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.