Single-Molecule DNA Sequencing with Engineered Nanopores

描述（由申请人提供）：在纳米孔链测序中，DNA的单链移动通过窄孔，并且碱基在它们通过阅读头时被识别。 在这里，我们专注于将α-溶血素（阿勒）蛋白纳米孔的链测序付诸实践所需的剩余任务。 纳米孔测序是一种快速的实时技术;它不需要耗时的试剂循环添加。 在实现了具有106个孔的芯片之后，我们预计到2014年，纳米孔测序将以非常短的样品制备时间实现15分钟的基因组测序。 此外，纳米孔测序将能够识别修饰的碱基并直接测序RNA。 在过去的四年中，我们已经取得了重大进展;我们已经表明，所有四个核碱基都可以在完整的DNA链中被识别，并证明了由DNA聚合酶驱动的实时单核苷酸链易位。 我们现在能够整合这些发现，并利用纳米孔阵列实现超快速测序。在下一个财政年度，我们将：1。通过使用阿勒纳米孔（通过常规诱变、非天然氨基酸诱变和靶向化学修饰工程化）来优化碱基识别，以产生适合于实时测序的DNA阅读头。 2.实现非酶DNA测序的链移位控制。 DNA移动的速度将通过使用轮烷来减慢，轮烷是由小分子或工程蛋白质环制成的，因此可以通过现有的记录技术检测碱基。 3.在一个平行的努力中，通过使用DNA聚合酶酶促控制DNA运动。 聚合酶也将用于两种新的测序模式，基于与核碱基掺入相关的构象变化的纳米孔检测。 4.开发含有多达106个阿勒纳米孔的芯片。 首先，将开发光学检测106芯片的原型。 第二，阿勒孔将被放置在已经用电子束钻入氮化硅膜的孔阵列中，从而完全避免使用脂质双层。 在第四年，纳米孔测序的这些关键方面将被集成到超快速测序设备中。 公共卫生相关性：超快速DNA测序，允许医生提供完整的现场基因组信息，将进一步推动临床实践，提供一个水平的护理，这将是令人难以置信的，就在几年前。 我们的目标是在未来四年内通过减少蛋白质纳米孔的超快速测序来为基因组医学的革命做出贡献。

DESCRIPTION (provided by applicant): In nanopore strand sequencing, a single strand of DNA moves through a narrow pore and the bases are identified as they pass a reading head. Here, we focus on the remaining tasks required to put into practice strand sequencing with the a-hemolysin (aHL) protein nanopore. Nanopore sequencing is a rapid real-time technology; it does not require the time-consuming cyclic addition of reagents. After implementing a chip with 106 pores, we expect nanopore sequencing to achieve a 15-minute genome by 2014 with a very short sample preparation time. In addition, nanopore sequencing will be able to identify modified bases and to sequence RNA directly. Over the past four years, we have made significant progress; we have shown that all four nucleobases can be identified within intact DNA strands and demonstrated real-time single- nucleotide strand translocation driven by DNA polymerase. We are now in a position to integrate these findings, and with a nanopore array, achieve ultrarapid sequencing. In the next funding period, we will: 1. Refine base recognition by using aHL nanopores, engineered by conventional mutagenesis, unnatural amino acid mutagenesis and targeted chemical modification, to produce DNA reading heads fit for real-time sequencing. 2. Achieve control of strand translocation for non-enzymatic DNA sequencing. The speed of DNA movement will be slowed by the use of rotaxanes, made from small molecules or engineered protein rings, so that bases can be detected by available recording techniques. 3. In a parallel effort, control DNA movement enzymatically by using DNA polymerase. The polymerase will also be employed in two novel sequencing modes, based on nanopore detection of conformational changes associated with nucleobase incorporation. 4. Develop chips containing up to 106 aHL nanopores. First, the prototype of an optically-detected 106-chip will be developed. Second, aHL pores will be placed in arrays of apertures that have been bored into a silicon nitride film with an electron beam, thereby avoiding the use of lipid bilayers altogether. In year 4, these crucial aspects of nanopore sequencing will be integrated into an ultrarapid sequencing device. PUBLIC HEALTH RELEVANCE: Ultrarapid DNA sequencing, allowing the physician to provide full on-the-spot genomic information, will further advance clinical practice to provide a level of care that would have been unbelievable just a few years ago. It is our goal to contribute to the revolution in genomic medicine by reducing ultrarapid sequencing with protein nanopores to practice over the next four years.