Single-Molecule DNA Sequencing with Engineered Nanopores

描述（由申请人提供）：在过去的五年中，已经克服了纳米孔测序中的重要技术问题，最终获得了具有显著优点的实用装置，包括对长度为100，000个碱基的DNA链进行测序的能力。然而，一个单独的纳米孔需要一年多的时间来测序人类基因组。为了在几分钟内完成一个基因组的测序，必须同时对数千个DNA进行测序。我们的提案通过开发生产和监测纳米孔测序阵列的新方法来解决这个问题。 我们将探讨三种形成数组的一般方法。首先，我们将研究涉及液滴界面双层（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，并将通过电或光记录进行监测。在后一种情况下，阵列中的每个双层将包含多功能纳米孔，从而允许数据采集速率的大幅增加。设计用于稳定阵列的新脂质化学将是这种方法的关键方面。第二，无双层系统将通过在薄的固态膜（特别是氮化硅）的孔中沉积蛋白质孔来制造。将开发用于氮化硅上的表面氧化物层的衍生化的新化学，以修改孔以容纳孔并防止孔与孔壁之间的电流泄漏。第三，DNA纳米结构将用于构建阵列。适用于测序应用的纳米孔将由DNA构建，以与DIB或固态阵列一起使用。DNA纳米孔或蛋白质纳米孔也将连接到DNA瓦片或支架上，所述DNA瓦片或支架被设计为保持适合于光学检测的孔到孔间距。最后，我们将研究与三类阵列兼容的核碱基检测技术。并行电检测的进步将被利用。还将探索旨在大大增加可以监测的孔隙数量的光学方法，包括通过使用较少的透镜宽场检测来增加视场的方法。更多的推测性超分辨率方法将被研究，以确定孔隙之间的间距是否可以减少到亚微米范围。 我们提出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含有104个，可能是106个或更多功能孔的芯片。这些芯片不仅可以提供1,000美元的基因组，而且可以在短短10分钟内提供超快速的基因组。

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.