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

描述（由申请人提供）：DNA的分离对于我们理解人类生物学和健康的进步至关重要。病原生物和微生物群落的基因组测序、基因分型、表型以及宏基因组分析都依赖于DNA分离方法，最常见的是基于DNA片段长度。然后必须对相同长度的片段进行测序或按序列分开。该提案的目标是发明一种微流控芯片，用于简单，快速，低成本地分离二维DNA：长度和序列。这将通过首先使用常规筛分聚合物按长度分离DNA，然后使用由鸟苷化合物自缔合形成的新凝胶介质在序列的第二维中分离相同长度的DNA片段来实现。这种鸟苷凝胶或“G-凝胶”显示出基于序列分离DNA的前景，而不需要由三维结构诱导的迁移率变化或对热/化学变性的抗性，这些是现有技术的基础。本论文的主要目的是：（1）利用毛细管电泳技术系统地研究G-凝胶对DNA序列的选择性，并评估基于序列的分辨率的极限;（2）设计并制作一个用于二维DNA分离的微流控芯片;（3）分别优化二维DNA的分离;（4）优化组合的2-D分离并评估分辨率的极限;（5）测试芯片分离已通过标准方法充分表征的微生物DNA样品的能力。最终目标是一个独立的集成设备，将二维分离与板载电源和检测集成到一个扩展的芯片中，该芯片将包括用于样品处理的预分析步骤，包括DNA提取，纯化和PCR。实现这样一个集成平台将为实时DNA分析提供一个真正的芯片实验室，这将为广泛的应用提供重要的优势，例如，在护理点和偏远地区或灾难现场等具有挑战性的环境中进行常规基因组分析，以及分析病原体和与健康相关的微生物群落并监测它们对环境压力的反应。

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.