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读取器，能够对线性化的长基因组材料进行单分子作图。预期的实施方案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的兆碱基片段，具有亚酶分辨率。此外，该芯片将容纳单个DNA分子的大规模并行分析，以允许沿着DNA的序列基序或多态性位点的标准化、高通量映射。 我们提出了一种用于单个DNA分子的条形码化策略（芯片、读取器和测定）。我们的条形码化策略是基于直接荧光成像和定位多个序列基序或多态性位点沿着线性化DNA分子。这种能力将改变生物学分析，允许高灵敏度检测基因座，用于全基因组关联研究，特别是需要单倍型信息的情况。此外，还可以进行病原体基因组的作图。该装置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，以便可以进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分子在大规模平行的纳米级通道中流动和线性化。纳米通道装置的优点是，由于通道内分子的物理限制，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，从而允许位点特异性荧光标记的空间映射。 该项目的完成将产生一个芯片，成像系统和分析映射序列基序和多态性位点沿沿着单分子的DNA与800 bp的分辨率。这样的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异的附加信息，例如拷贝数变异和易位。

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.