99.99% Accuracy Direct DNA Sequencing via the Protein Nanopore Method

描述（由申请人提供）：我们的目的是开发一种基于跨膜蛋白孔α-溶血素（阿勒）中离子电流阻断的DNA测序装置。与其他提出的基于纳米孔的方法相比，蛋白质孔电流阻断（PPCB）方法在概念上和技术上都可以说是最简单的，但最近已经被更复杂的方法所取代。最近的电子读出和双层脂质膜的进步，大大减轻了先前的信噪比和鲁棒性的问题。新的实验数据和a-HL内DNA运动的精确随机模型（SMDM）现在表明通过PPCB方法进行测序的阈值可行性。对已知输入DNA序列的计算SMDM响应的检查表明，通过PPCB方法测序的主要问题是碱基通过孔的顺序的随机变化，导致三个可量化的误差来源。该程序将对本装置进行特定的结构和测量参数修改，以减少每种类型的误差并产生最小的总测序误差。将通过对高达30 M碱基的天然DNA进行测序来评价最终装置。 公共卫生相关性：该项目提供了一条低成本DNA测序的途径。确定DNA序列在研究基本生物过程的基础研究以及诊断或法医研究等应用领域中是有用的。DNA测序的出现大大加速了生物学研究和发现，但目前的方法复杂且昂贵，因此限制了它们的适用性。简单、便宜的DNA测序提供了将该过程的优点应用于日常医疗和法医应用的能力。

DESCRIPTION (provided by applicant): Our aim is to develop a DNA sequencing device based upon the blockade of ionic current in the transmembrane protein pore a-hemolysin (aHL). Compared to other proposed nanopore-based approaches, the protein pore current blockade (PPCB) method is arguably the simplest, both conceptually and technologically, but recently has been passed over in favor of more complex methods. Recent electronic readout and bilayer lipid membrane advances by the proposers have greatly alleviated prior signal-to-noise ratio and robustness issues. New experimental data and an accurate Stochastic Model for DNA Motion (SMDM) within a-HL now indicate threshold feasibility for sequencing by the PPCB method. Inspection of calculated SMDM responses to known input DNA sequences shows that the principal issue for sequencing via the PPCB method is random variance in the order the bases pass through the pore, leading to three quantifiable sources of error. This program will make specific structural and measurement parameter modifications to the present apparatus to reduce each type of error and to produce a minimal overall sequencing error. The final apparatus will be evaluated by sequencing up to 30 Mbases of natural DNA. PUBLIC HEALTH RELEVANCE: This project offers a path to low cost DNA sequencing. Determining the DNA sequence is useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery, but current methods are complicated and expensive, thus limiting their applicability. Simple, inexpensive DNA sequencing offers the capability to apply the benefits of the process to everyday medical and forensic applications.