Nanofluidic Platforms for High Resolution Mapping of Genomic DNA

描述(申请人提供)：我们建议开发一个用于高通量全基因组限制图谱的纳米流体平台。该平台包括用于从细胞中提取染色体DNA的单片集成流体，其在长纳米通道中被限制性内切酶消化，以及有序限制片段的高分辨率测定。与其他基于纳米通道的方法不同，我们不依赖于高度受限和拉长的DNA分子的成像。相反，荧光标记的DNA被相对较大直径(300-500 nm)通道中的限制性内切酶消化，在该通道中，纳米通道的限制和静电力阻止了相邻片段的扩散混合。消化后，有序的片段被电泳驱动到进样点，在那里通道直径减小到~100 nm。当每个碎片到达这一点时，它会被更高的电场加速，从而与其后面的相邻碎片产生分离。这些分离的碎片通过聚焦的激光光斑迁移，并检测和分析每个荧光脉冲的持续时间和积分强度，以确定碎片大小。我们预计我们的方法有几个优势；它依赖于大于100 nm的纳米通道，确保在原型制作后，可以使用低成本、高通量的方法制造器件。制造直径为300-500 nm的超长纳米通道的能力使我们能够将长基因组DNA限制在反应纳米通道中，大大减少了从较小的DNA分子组装MAP的需要。在芯片上集成DNA提取增加了询问完整染色体DNA的可能性，完全消除了MAP组装，并提供了真正的全球覆盖。单点检测消除了对图像存储和分析的需要。此外，还可能提供许多独特的映射前或映射后功能机会。我们已经进行了初步的研究，展示了将来自反应纳米通道的片段有序地注入较小的检测纳米通道中，以及这种方法解析相邻片段的能力。连同Ramsey小组和其他人开发的其他要素，我们相信我们将在拟议项目的前半部分生成限制图。因此，我们的团队包括在下一代测序(NGS)和生物信息学方面具有专业知识的成员。我们将对照参考序列和使用其他方法生成的物理图谱来验证我们的限制图。我们还将展示我们的限制图作为从头组装NGS数据的脚手架的用途。从这些评估中确定的任何错误或偏见都将指导平台改进。

DESCRIPTION (provided by applicant): We propose to develop a nanofluidic platform for the high-throughput restriction mapping of complete genomes. This platform consists of monolithically integrated fluidics for the extraction of chromosomal DNA from cells, its digestion by restriction endonucleases in a long nanochannel, and the high-resolution sizing of ordered restriction fragments. In contrast to other nanochannel-based approaches, we do not rely on the imaging of highly confined and elongated DNA molecules. Rather, fluorescently labeled DNA is digested by restriction endonucleases in a relatively large diameter (300 - 500 nm) channel in which nanochannel confinement and electrostatic forces prevent diffusive mixing of adjacent fragments. After digestion, the ordered fragments are electrophoretically driven to an injection point where the channel diameter decreases to ~100 nm. As each fragment reaches this point, it is accelerated by the higher electric field, creating separation from its trailing neighbors. Thse separated fragments migrate through a focused laser spot and the duration and integrated intensity of each fluorescence pulse is detected and analyzed to determine fragment size. We anticipate several advantages to our approach; it relies on nanochannels greater than 100 nm, ensuring that, after prototyping, devices can be fabricated using low-cost, high-throughput methods. The ability to fabricate very long nanochannels with diameters of 300 - 500 nm enables us to confine long genomic DNA in the reaction nanochannel, greatly reducing the need for map assembly from smaller DNA molecules. Integration of DNA extraction on chip increases the probability of interrogating intact chromosomal DNA, eliminating map assembly entirely and providing truly global coverage. Single-point detection obviates the need for image storage and analysis. In addition, many unique opportunities for pre- or post-mapping functionality are possible. We have conducted preliminary studies demonstrating the ordered injection of fragments from a reaction nanochannel into a smaller detection nanochannel and the ability of this approach to resolve neighboring fragments. Together with other elements developed by the Ramsey group and others, we believe that we would be generating restriction maps during the first half of the proposed project. Consequently, our team includes members with expertise in next generation sequencing (NGS) and bioinformatics. We will validate our restriction maps against reference sequences and physical maps generated using other methods. We will also demonstrate the utility of our restriction maps as scaffolds for de novo assembly of NGS data. Any errors or biases determined from these assessments will direct platform improvements.