High resolution analysis of linear genomic DNA in Parallel nanochannel arrays

描述(由申请人提供)： 我们正在开发一种纳米芯片设备，用于操作长基因组DNA，用于高分辨率(千碱基)、癌症生物标记物的全基因组分析，如基因扩增、缺失和易位。这些染色体结构异常与癌变过程密切相关，是许多类型癌症的重要诊断、预后和治疗指标。尽管聚合酶链式反应为检测和分析这些异常提供了最终的(单碱基)分辨率，但要以全面、线性的方式扫描整个基因组是不切实际的。依赖于探测染色体的技术，如中期FISH，虽然提供了泛基因组图，但无法解析Mb范围以下的结构。通过探测未压缩的间期DNA，可以提高分辨率，但会丢失基因组的空间组织，因此难以获得多重和定量的信息。通过伸展(线性化)间期DNA，使用“分子梳理”或“光学作图”等技术，有可能以空间上有意义的方式探测特定的基因座，并且分辨率在kb范围内。然而，机械线性化DNA的技术本身是可变的，导致分子不一致的拉伸，分子往往会交叉并自行收缩。这使得生物医学社区的高通量方法等技术很难标准化。我们正在开发一种创新的替代DNA机械拉伸的方法。我们已经发现，由于DNA聚合物的自我回避性质，单个DNA分子在流入限制纳米级通道(纳米通道)时，将以一致的方式伸长和拉直。我们已经使用了一种新的纳米压印光刻技术来在硅芯片中可靠地制造纳米通道结构，并证明了这些纳米通道中的DNA可以被可视化并测量其尺寸。我们现在要问的问题是，我们能否用位点特异性的探针来定量询问这种线性化的DNA，以检测与癌症相关的染色体结构异常？我们的产品，纳米通道阵列芯片，将包括一个集成平台的一部分，用于常规和标准化的DNA结构定量分析，使数据存档和跨实验室比较成为可能。

DESCRIPTION (provided by applicant): We are developing a nanochip device for manipulating long genomic DNA for high-resolution (kilobase), whole-genome analysis of cancer biomarkers such as gene amplifications, deletions, and translocations. These chromosome structural aberrations are strongly implicated in the process of malignant transformation and are important diagnostic, prognostic, and therapeutic indicators for many types of cancer. Although PCR offers the ultimate (single-base) resolution for detecting and analyzing these anomalies, it is impractical for scanning the entire genome in a comprehensive, linear fashion. Techniques that rely on probing chromosomes, such as metaphase FISH, while providing a pan-genomic view, cannot resolve structures below the Mb range. By probing uncompressed interphase DNA, resolution can be improved, but spatial organization of the genome is lost, so multiplexed and quantitative information is difficult to obtain. By stretching out (linearizing) interphase DNA, using techniques such as "molecular combing" or "optical mapping," it is possible to probe specific loci in a spatially-significant way, and with resolutions in the kb range. However, techniques for mechanically linearizing DNA are inherently variable, leading to inconsistent stretching of molecules, which often cross over and retract upon themselves. This makes it difficult to standardize such techniques as high throughput methods for the biomedical community. We are developing an innovative alternative to mechanical stretching of DNA. We have found that individual DNA molecules, because of the self-avoiding nature of the DNA polymer, will elongate and straighten in a consistent manner when streamed into confining nanometer-scale channels (nanochannels). We have used a novel nanoimprint lithography technique to reliably manufacture nanochannel structures in silicon chips and have demonstrated that DNA in these nanochannels can be visualized and their dimensions measured. We now ask the question, can we quantitatively interrogate this linearized DNA with locus-specific probes for the detection of chromosome structural aberrations associated with cancer? Our product, the nanochannel array chip, will comprise part of an integrated platform for the routine and standardized quantitative analysis of DNA structure that will enable archiving and cross-laboratory comparison of data.