Nanochannel Devices for High Throughput Single Molecule DNA mapping and Haplotypi

描述（由申请人提供）：本提案的长期目标是开发一种完全集成的纳米通道芯片和阅读器，能够对线性化的长基因组材料进行单分子定位。预期的实施例将允许以亚千碱基分辨率直接从样品（可能是单个细胞）中提取的DNA的百万碱基片段的直接可视化和分析。此外，该芯片将容纳单个DNA分子的大规模并行分析，以允许沿DNA的序列基序或多态位点的标准化，高通量映射。我们提出了一种针对单个DNA分子的条形码策略（芯片，读取器和分析）。我们的条形码策略是基于直接荧光成像和沿线性化DNA分子的多个序列基序或多态位点的定位。这种能力将改变生物学分析，允许高度敏感的基因位点检测，用于全基因组关联研究，特别是在需要单倍型信息的地方。此外，还可以绘制病原体基因组图谱。该设备商业化的一个关键考虑因素是单个DNA分子的一致线性化和成像，从而可以执行标记位点的高分辨率映射。鉴于这一要求，我们提出了一种纳米流体装置，其中单个DNA分子在大规模平行纳米级通道中流动和线性化。纳米通道器件的优点是，由于分子在通道内的物理限制，DNA可以以标准化和可重复的方式线性化。在线性化过程中，使用高分辨率显微镜系统对DNA进行荧光成像，从而允许对特定位点的荧光标记进行空间映射。该项目的完成将产生一个芯片、成像系统和测定，用于沿着DNA单分子绘制序列基序和多态位点，分辨率为800 bp。这种设备将通过提供关键的单倍型信息来改变和增强我们对遗传疾病的理解。该装置可以进一步扩展以提供关于基因组中的结构变化的附加信息，例如拷贝数变化和易位。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to develop a fully integrated nanochannel chip and reader capable of single molecule mapping of linearized, long 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 sub-kilobase resolution. Furthermore, the chip will accommodate massively parallel analyses of individual DNA molecules to permit standardized, high-throughput mapping of sequence motifs or polymorphic sites along the DNA. We propose a barcoding strategy (chip, reader and assay) for single DNA molecules. Our barcoding strategy is based on direct fluorescent imaging and localization of multiple sequence motifs or polymorphic sites along a linearized DNA molecule. Such capabilities will transform biological analyses, permitting highly sensitive detection of genetic loci for genome wide association studies, especially where haplotype information is required. In addition, mapping of pathogen genomes can also be performed. A critical consideration for the commercialization of this device is the consistent linearization and imaging of individual DNA molecules such that high resolution mapping of labeled sites can be performed. In light of this requirement, we propose a nanofluidic device in which individual DNA molecules are streamed and linearized in massively parallel nanoscale channels. The advantage of a nanochannel device is that DNA can be linearized in a standardized and repeatable manner due to the physical confinement of molecules within the channel. During linearization, the DNA is fluorescently imaged using a high resolution microscopy system thus permitting spatial mapping of site-specific fluorescent labels. Completion of this project will result in a chip, imaging system and assay for mapping sequence motifs and polymorphic sites along single molecules of DNA with 800 bp resolution. Such a device will transform and enhance our understanding of genetic diseases by providing crucial haplotype information. The device can be further extended to provide additional information regarding structural variations in the genome such as copy number variations and translocations.