Single-Molecule DNA Sequencing with Engineered Nanopores

描述(申请人提供)：在纳米孔链测序中，一条DNA单链通过一个狭窄的小孔，当它们通过读数头时，碱基被识别。在这里，我们将重点放在用a-溶血素(AHL)蛋白纳米孔进行链测序所需的剩余任务上。纳米孔测序是一种快速实时技术；它不需要耗时的试剂循环添加。在实施了一个有106个孔的芯片后，我们预计纳米孔测序将在2014年前以非常短的样本准备时间实现15分钟的基因组。此外，纳米孔测序将能够识别修饰的碱基并直接对RNA进行测序。 在过去的四年里，我们取得了重大进展；我们已经证明，在完整的DNA链中可以识别所有四个核苷酸碱基，并证明了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测序。在下一个资助期，我们将：1.利用AHL纳米孔，通过常规诱变、非天然氨基酸诱变和靶向化学修饰来改进碱基识别，以生产适合实时测序的DNA读数头。2.实现了非酶DNA测序的链转位控制。DNA移动的速度将通过使用由小分子或工程蛋白质环制成的轮烷来减缓，这样就可以通过现有的记录技术来检测碱基。3.同时，利用DNA聚合酶控制DNA的移动。该聚合酶还将用于两种新的测序模式，基于纳米孔检测与碱基掺入相关的构象变化。4.开发包含多达106个AHL纳米孔的芯片。首先，将开发一种光学检测106芯片的原型。其次，AHL小孔将被放置在用电子束钻入氮化硅薄膜的孔阵列中，从而完全避免使用脂类双层。在第4年，纳米孔测序的这些关键方面将被集成到一个超快测序设备中。 与公共卫生相关：超快速DNA测序使医生能够提供完整的现场基因组信息，这将进一步推动临床实践，提供几年前还无法相信的护理水平。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测序来为基因组医学革命做出贡献。

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.