Nanopore sequencing of DNA with MspA

描述（由申请人提供）：本更新提案的目的是实现MspA纳米孔测序技术。通过该计划的资金，我们的团队专门针对纳米孔测序设计了孔蛋白MspA。通过将MspA与进行性酶技术相结合，我们的团队展示了二十年前首次设想的纳米孔测序的愿景目标。 在持续的资金支持下，我们将采取必要的措施，为工业整合准备纳米孔测序。我们还将探索使纳米孔测序成为更强大工具的改进和变化。 我们的具体目标是：（1）开发我们目前的纳米孔测序过程所必需的碱基识别算法，（2）通过合理和大胆的基因工程进一步精制孔蛋白MspA，以优化其测序质量，（3）通过降低其随机特性来理解和改善DNA易位动力学，以及（4）展示天然基因组DNA的长片段的测序，包括鉴定表观遗传修饰。 迄今为止，我们团队的成功使我们能够建立合作伙伴关系，并获得许多优秀实验室的支持，这些实验室的专业知识帮助我们推进纳米孔测序。我们将与我们的合作伙伴实验室合作，以完成本提案中概述的目标。 我们的目标是通过提供一种技术来改善人类的生活质量，该技术可以以低于1000美元的价格准确地对人类基因组进行测序，并在临床实践所需的短时间内提供测序结果。

DESCRIPTION (provided by applicant): The objective of this renewal proposal is to bring nanopore sequencing technology with MspA to fruition. With funding through this program, our group has engineered the porin MspA specifically for nanopore sequencing. By combining MspA with a processive enzyme technology our team demonstrated the visionary goal of nanopore sequencing first conceived two decades ago. With continued funding, we will take the necessary steps to ready nanopore sequencing for industrial integration. We will also explore improvements and variations that make nanopore sequencing an even more powerful tool. Our specific aims are: (1) develop the base calling algorithms necessary for our present nanopore sequencing process, (2) further refine the porin MspA through rational - and bold - genetic engineering in order to optimize its sequencing quality, (3) understand and improve DNA translocation dynamics by reducing its stochastic character and (4) demonstrate sequencing of long segments of native genomic DNA including identification of epigenetic modifications. Our team's success to date has enabled us to form partnerships and gain support from many excellent labs whose expertise has assisted us to move nanopore sequencing forward. We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering a technique that can accurately sequence a human genome for well under $1000 and provide sequencing results within the short timeframe required for clinical practice.