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

用于按片段长度快速分离 DNA 的二维微流控平台

基本信息

批准号：
8717690
负责人：
Linda B. Mc GOWN
金额：
$ 19.14万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2016-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：DNA的分离对于我们对人类生物学和健康的理解的进步至关重要。基因组测序，基因分型，表型以及致病生物和微生物群落的宏基因组分析都依赖于DNA分离的方法，最常见的是基于DNA片段长度。然后，必须按序列对相同的片段进行测序或分离。该提案的目的是发明一个微流体芯片，以在二维中简单，快速，低成本的DNA分离：长度和序列。这将通过使用常规的筛分聚合物首先按长度分离DNA，然后在序列的第二维中分离出相同的DNA片段，并使用新的凝胶培养基在序列的第二维中分离，该介质是由鸟苷化合物自相关形成的新凝胶培养基。这种鸟嘌呤凝胶或“ G-gel”显示了根据序列分离DNA的希望，而无需由三维结构或对热/化学变性的抗性引起的迁移率转移，这是现有技术的基础。该建议的具体目的是：（1）使用毛细管凝胶电泳系统地研究G-凝胶的DNA序列选择性，并评估基于序列的分辨率的限制；（2）设计和制造微流体芯片，以进行二维（2-D）DNA分离；（3）在两个维度中的每个维度中分别优化分离；（4）优化组合的2-D分离并评估分辨率的限制；（5）测试芯片分离已完全表征标准方法的微生物DNA样品的能力。最终目标是一个独立的集成装置，将2-D分隔纳入板载电源和检测到扩展的芯片中，其中包括用于样品处理的预动作步骤，包括DNA提取，纯化和PCR。实现这种集成平台将为实时DNA分析提供真正的实验室芯片，该实验室将为广泛的应用提供重要的优势，例如，在护理点和远程或灾难网站（例如远程或灾难网站）中的常规基因组分析，以及针对病原体和健康相关的微生物社区和与环境压力的概述。