Two-Dimensional Microfluidic Platform for Rapid DNA Separation by Fragment Length

描述(由申请者提供)：DNA的分离对于我们对人类生物学和健康的理解起着至关重要的作用。病原生物体和微生物群落的基因组测序、基因分型、表型分析以及元基因组分析都依赖于DNA分离的方法，最常见的方法是DNA片段长度。然后，必须对相同长度的片段进行测序或按顺序分离。这项提议的目标是发明一种微流控芯片，用于从长度和序列两个维度简单、快速、低成本地分离DNA。这将通过首先使用传统的筛分聚合物按长度分离DNA，然后使用由鸟苷化合物自结合形成的新的凝胶介质在序列的第二维分离相同长度的DNA片段来实现。这种鸟苷凝胶，或“G-凝胶”，显示了根据序列分离DNA的前景，而不需要由于三维结构或对热/化学变性的抵抗而引起的迁移率变化，而这些是现有技术的基础。这项建议的具体目的是：(1)使用毛细管凝胶电泳法系统地研究G-Gel的DNA序列选择性，并评估基于序列的分辨率的限度；(2)设计和制造用于二维(2D)DNA分离的微流控芯片；(3)分别在两个维度上分别优化分离；(4)优化组合的二维分离并评估分辨率限度；(5)测试芯片分离已通过标准方法充分表征的微生物DNA样本的能力。最终目标是一种自给自足的集成设备，将带有车载电源和检测的二维分离功能整合到一个扩展的芯片中，该芯片将包括样本处理的预分析步骤，包括DNA提取、纯化和聚合酶链式反应。这种综合平台的实现将为实时DNA分析提供真正的芯片上实验室，这将为广泛的应用提供重要的优势，例如在护理地点和偏远地区或灾害地点等具有挑战性的环境中进行常规基因组分析，以及描绘病原体和与健康有关的微生物群落并监测它们对环境应激源的反应。

DESCRIPTION (provided by applicant): Separation of DNA has been central to advances in our understanding of human biology and health. Genome sequencing, genotyping, phenotyping as well as metagenomic analysis of pathogenic organisms and microbial communities have all relied on methods for DNA separation, most commonly based on DNA fragment length. Same-length fragments must then be sequenced or separated by sequence. The goal of this proposal is to invent a microfluidic chip for simple, rapid, low-cost separation of DNA in two dimensions: length and sequence. This will be achieved by first separating DNA by length using conventional sieving polymers and then separating the same-length DNA fragments in the second dimension of sequence using a new gel medium that is formed by self-association of guanosine compounds. This guanosine gel, or "G-gel", shows promise for separation of DNA on the basis of sequence without the need for mobility shifts induced by three-dimensional structure or resistance to thermal/chemical denaturation that are the basis for existing techniques. The specific aims of this proposal are to: (1) Use capillary gel electrophoresis to systematically investigate the DNA sequence selectivity of the G-gels and evaluate limits of sequence-based resolution; (2) Design and fabricate a microfluidic chip for the two-dimensional (2-D) DNA separation; (3) Individually optimize separations in each of the two dimensions; (4) optimize the combined, 2-D separation and evaluate limits of resolution; (5) test the ability of the chip to separate microbial DNA samples that have been fully characterized by standard methods. The ultimate goal is a self-contained, integrated device that would incorporate the 2-D separation with on-board power supply and detection into an expanded chip that would include pre-analysis steps for sample processing, including DNA extraction, purification and PCR. Realization of such an integrated platform would provide a true lab-on-a-chip for real-time DNA analysis that would offer important advantages for wide-ranging applications, for example, routine genomic analysis at point-of-care and in challenging settings such as remote areas or disaster sites, and for profiling pathogenic organisms and health-related microbial communities and monitoring their responses to environmental stressors.