Singer Alon, McNally Ben, Torre Ruby Dela, Meller Amit
Department of Biomedical Engineering, Boston University, Boston, MA, USA.
Methods Mol Biol. 2012;870:99-114. doi: 10.1007/978-1-61779-773-6_6.
Nanopore-based DNA analysis is an extremely attractive area of research due to the simplicity of the method, and the ability to not only probe individual molecules, but also to detect very small amounts of genomic material. Here, we describe the materials and methods of a novel, nanopore-based, single-molecule DNA sequencing system that utilizes optical detection. We convert target DNA according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of photon bursts that can be detected with a custom-made microscope. We do not use any enzymes in the readout stage; thus, our system is not limited by the highly variable processivity, lifetime, and inaccuracy of individual enzymes that can hinder throughput and reliability. Furthermore, because our system uses purely optical readout, we can take advantage of high-end, wide-field imaging devices to record from multiple nanopores simultaneously. This allows an extremely straightforward parallelization of our system to nanopore arrays.
基于纳米孔的DNA分析是一个极具吸引力的研究领域，这得益于该方法的简单性，以及不仅能够探测单个分子，还能检测极少量基因组材料的能力。在此，我们描述了一种新型的、基于纳米孔的单分子DNA测序系统的材料和方法，该系统利用光学检测。我们根据二进制编码对目标DNA进行转换，该编码由带有两种荧光团的分子信标识别。然后使用固态纳米孔依次剥离信标，产生一系列光子爆发，可通过定制显微镜进行检测。我们在读出阶段不使用任何酶；因此，我们的系统不受单个酶的高变率、寿命和不准确性的限制，这些因素会阻碍通量和可靠性。此外，由于我们的系统使用纯光学读出，我们可以利用高端宽场成像设备同时记录多个纳米孔的数据。这使得我们的系统能够极其直接地并行到纳米孔阵列。
