Single-Molecule DNA Sequencing with Engineered Nanopores

描述(由申请人提供)：在过去的五年中，纳米孔测序中的重要技术问题已经被攻克，最终形成了一种具有显著优势的可行设备，包括能够对100,000个碱基的DNA链进行测序。然而，单个纳米孔需要一年多的时间才能对人类基因组进行测序。要在几分钟内对一个基因组进行测序，对数千个DNA进行并行测序是至关重要的。我们的建议通过开发新的方法来生产和监测纳米孔测序阵列来解决这个问题。我们将探索形成数组的三种一般方法。首先，我们将检查涉及液滴界面双层(DIB)的阵列。DIB阵列将基于水-水、水-水凝胶或水凝胶-水凝胶界面，并将通过电子或光学记录进行监测。在后一种情况下，阵列中的每个双层都将包含多个功能纳米孔，从而大幅提高数据采集的速度。设计用于稳定阵列的新的脂类化学将是这一方法的关键方面。其次，无双层系统将通过在薄膜固态薄膜(特别是氮化硅)的孔洞中沉积蛋白质孔来制造。将开发用于氮化硅表面氧化层衍生化的新化学方法，以修改孔以适应孔并防止孔和孔壁之间的电流泄漏。第三，DNA纳米结构将被用于构建阵列。适用于测序应用的纳米孔将由DNA构建，用于DIB或固态阵列。DNA纳米孔或蛋白质纳米孔也将附着在DNA瓷砖或支架上，以保持适合光学检测的孔与孔之间的间距。最后，我们将研究与这三类阵列兼容的碱基检测技术。将利用并行电检测方面的进展。还将探索旨在大幅增加可监测气孔数量的光学方法，包括通过使用无透镜广域探测来增加视场的方法。将研究更具推测性的超分辨率方法，以确定是否可以将孔间距减小到亚�m范围。我们建议的研究建立在强大的初步数据和我们在化学和化学生物学方面的专业知识的基础上，以开发新的方法来推进大规模并行纳米孔测序。这里提出的测序技术有望提供包含104个，甚至可能106个或更多功能孔的芯片。这些芯片不仅可以提供1000美元的基因组，还可以在短短10分钟内提供超快的基因组。

DESCRIPTION (provided by applicant): Important technical problems in nanopore sequencing have been overcome within the last five years, culminating in a practicable device with significant advantages, including the ability to sequence DNA strands 100,000 bases in length. Nevertheless, an individual nanopore would take more than a year to sequence a human genome. To sequence a genome in minutes, it is essential to sequence many thousands of DNAs in parallel. Our proposal addresses that issue by developing new methods to produce and monitor nanopore sequencing arrays. We will explore three general means to form arrays. First, we will examine arrays involving droplet interface bilayers (DIB). DIB arrays will be based on aqueous-aqueous, aqueous-hydrogel or hydrogel- hydrogel interfaces, and will be monitored by electrical or optical recording. In the latter case, each bilayer in the array will contain multple functional nanopores allowing a substantial increase in the rate of data acquisition. New lipid chemistry designed to stabilize the arrays will be a critical aspect of this approach. Second, bilayer-free systems will be fabricated by depositing protein pores in apertures in thin solid-stat films, notably silicon nitride. New chemistry for the derivatization of the surface oxide layer on silicon nitride will be developed to modify the apertures to accommodate the pores and to prevent current leaks between the pores and the aperture walls. Third, DNA nanostructures will be employed to build arrays. Nanopores suitable for sequencing applications will be constructed from DNA for use with either DIB or solid-state arrays. DNA nanopores or protein nanopores will also be attached to DNA tiles or scaffolds designed to maintain a pore-to-pore spacing suitable for optical detection. Finally, we will investigate nucleobase detection techniques compatible with the three classes of arrays. Advances in parallel electrical detection will be exploited. Optical approaches designed to greatly increase the number of pores that can be monitored will also be explored, including means to increase the field of view by using lens less wide-field detection. More speculatively super-resolution approaches will be investigated to determine whether the spacing between pores can be decreased into the sub-�m range. Our proposed studies build on strong preliminary data and our expertise in chemistry and chemical biology to develop new approaches to advance massively parallel nanopore sequencing. The sequencing technologies proposed here promise to deliver chips containing 104, and possibly 106 or more, functional pores. These chips will deliver not only a $1,000 genome, but an ultra-rapid genome in as little as 10 minutes.