99.99% Accuracy Direct DNA Sequencing via the Protein Nanopore Method

描述（由申请人提供）：我们的目标是开发一种基于跨膜蛋白孔a-溶血素（aHL）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展大大缓解了先前的信噪比和鲁棒性问题。新的实验数据和精确的a-HL DNA运动随机模型（SMDM）现在表明PPCB方法测序的阈值可行性。对已知输入DNA序列计算的SMDM响应的检验表明，通过PPCB方法进行测序的主要问题是碱基通过孔的顺序随机变化，导致三个可量化的误差来源。该程序将对现有仪器进行具体的结构和测量参数修改，以减少每种类型的误差，并产生最小的总体测序误差。最终的仪器将通过测序多达30个碱基的天然DNA来评估。

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.