Single-Molecule DNA Sequencing with Engineered Nanopores

描述（由申请人提供）：在纳米孔链测序中，单链DNA通过一个狭窄的孔，碱基在通过读取头时被识别。在这里，我们将重点放在使用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的技术；它不需要耗时的循环添加试剂。在实现了具有106个孔的芯片之后，我们预计到2014年，纳米孔测序将在非常短的样品制备时间内实现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对RNA进行测序。

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.