Hybrid nanochannel arrays for single molecule linear, genome analysis

描述(由申请人提供)：项目摘要/摘要：这项建议的长期目标是开发一种完全集成的芯片和读取器，能够对线性化的自然状态基因组材料进行单分子分析。预期的实施例将允许直接可视化和分析直接从样品(可能是单个细胞)中提取的DNA的百万碱基片段，分辨率优于400BP。此外，该芯片将以高通量方式容纳对单个DNA分子的大规模并行分析，从而及时提供统计上的相关数据。我们的方法将集中在纳米通道阵列的开发上，这是一种我们以前演示过的以大规模并行方式线性化DNA单分子的设备。该设备依赖于直径小于100 nm的通道阵列。当DNA通过这样的通道流动时，它会受到物理上的限制，被迫解开，从而允许使用荧光显微镜进行线性询问。这样的分析可以极大地增强我们对与癌症和其他遗传性疾病相关的基因组不稳定性的理解。这种器件商业化的一个关键考虑因素是纳米通道和支持芯片体系结构的设计和制造。目前，该器件由硅组成，并使用纳米压印光刻(NIL)进行图案化。NIL是为半导体工业开发的一种低成本、高产量的纳米制造工艺，作为进一步缩小传统集成电路中晶体管尺寸的手段。我们已经利用这项技术来制造生物芯片。虽然这一过程非常适合于创造纳米级的特征，但需要进一步开发，以适应直接从生物学家的吸管中提取的样本。我们假设，理想的器件将需要一个从宏观溶液环境到纳米级沟道的过渡区。此外，我们预计，通过依靠传统的、基于塑料的微流体制造技术来实现微米级的特征，并依靠基于硅的方法来实现亚微米级的过渡，从而实现通道本身的亚微米过渡，这种过渡是最经济的。这两个制造体制的整合至关重要。该项目的完成将产生一个单一的、集成的生物芯片设备，能够提供具有统计意义的、具有400BP分辨率的百万数据库DNA的单分子分析。这样的装置将对未来的单细胞、单分子分析至关重要。首席研究员/项目主任(最后、第一、中间)：曹、韩叙述：通过提供关于遗传多态性质的上下文信息，对长的基因组DNA进行单分子分析将使人们能够更好地理解和改进遗传疾病，特别是癌症的治疗。一个基于平行纳米通道的标准化平台可以作为未来患者护理中一致的、高通量基因组分析的基础。

DESCRIPTION (provided by applicant): Project Summary/Abstract: The long term objective of this proposal is to develop a fully integrated chip and reader capable of single molecule analysis of linearized, native state genomic material. The anticipated embodiment will permit direct visualization and analysis of megabase fragments of DNA extracted directly from a sample (possibly a single cell) with resolution better than 400 bp. Furthermore, the chip will accommodate massively parallel analyses of individual DNA molecules in a high-throughput manner thus providing statistically relevant data in a timely fashion. Our approach will focus on development of the nanochannel array, a device previously demonstrated by us to linearize single molecules of DNA in a massively parallel fashion. The device relies upon an array of channels with diameters less than 100 nm. As DNA is flowed through such channels, it becomes physically constrained and is forced to uncoil, thus permitting linear interrogation using fluorescent microscopy. Such analyses could serve to greatly enhance our understanding of genomic instabilities related to cancer and other genetic diseases. A critical consideration for the commercialization of this device is the design and manufacture of the nanochannels and supporting chip architecture. Currently, the device is composed of silicon and patterned using nanoimprint lithography (NIL). NIL is a low cost, high throughput nanomanufacturing process developed for the semiconductor industry as a means of further reducing the size of transistors in traditional integrated circuits. We have leveraged this technology for the manufacture of bio chips. Though well-suited for creating nanoscale features, further development of the process is required to accommodate a sample derived directly from the biologist's pipette. We hypothesize that the ideal device will require a transitional region from a macroscopic solution environment to the nanoscale channels. Furthermore, we expect that this transition is most economically developed by relying on conventional, plastic-based microfluidic manufacturing technologies for micron-scale features and silicon based approaches for the sub-micron transition leading to the channels themselves. Integration of these 2 manufacturing regimes is of critical importance. Completion of this project will result in a single, integrated biochip device capable of providing statistically significant, single molecule analysis of megabase DNA with 400 bp resolution. Such a device will be crucial to the future of single cell, single molecule analyses. Principal Investigator/Program Director (Last, First, Middle): Cao, Han Narrative: Single molecule analysis of long, genomic DNA will enable greater understanding and improved treatment of genetic diseases, especially cancer, by providing contextual information as to the nature of genetic polymorphisms. A standardized platform based on parallel nanochannels could serve as a basis for consistent, high-throughput genomic analyses in future patient care.