Nanopore sequencing of DNA with MspA

描述（由申请人提供）：本更新提案的目标是将MspA纳米孔测序技术付诸实践。在这个项目的资助下，我们的团队设计了专门用于纳米孔测序的孔蛋白MspA。通过将MspA与过程酶技术相结合，我们的团队展示了20年前首次构想的纳米孔测序的远景目标。有了持续的资金，我们将采取必要的步骤，为工业整合准备纳米孔测序。我们还将探索改进和变化，使纳米孔测序成为一个更强大的工具。我们的具体目标是：(1)为我们目前的纳米孔测序过程开发必要的碱基调用算法；(2)通过合理和大胆的基因工程进一步完善孔蛋白MspA，以优化其测序质量；(3)通过减少其随机特征来理解和改善DNA易位动力学；(4)展示原生基因组DNA长片段的测序，包括表观遗传修饰的鉴定。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，他们的专业知识帮助我们推动纳米孔测序向前发展。我们将与我们的合作伙伴实验室一起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提高人类生活质量，这种技术可以在1000美元以下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.