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Single-Molecule DNA Sequencing with Engineered Nanopores

使用工程化纳米孔进行单分子 DNA 测序

基本信息

批准号：
8499385
负责人：
M. Reza Ghadiri
金额：
$ 114.47万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8499385
关键词：
Amino Acids Caring Chemicals Chemistry Computer Analysis Cyclic Peptides Cyclodextrins DNA DNA Sequence DNA-Directed DNA Polymerase Detection Development Devices Electron Beam Engineering Epigenetic Process Exonuclease Film Fluorescence Funding Genetic Engineering Genome Genomics Goals Head Hemolysin Hybrids Hydrogen Bonding Individual Investigation Ions Length Lipid Bilayers Lipids Medicine Modification Molecular Monitor Movement Mutagenesis Nucleotides Physicians Polymerase Polymers Pore Proteins Positioning Attribute Preparation Protein Engineering Proteins RNA RNA Sequences Reading Reagent Rotaxanes Sampling Single-Stranded DNA Speed Spottings Surface Techniques Technology Testing Time Total Internal Reflection Fluorescent base clinical practice design divalent metal genome sequencing improved nanopore novel nucleic acid structure nucleobase polypeptide prototype public health relevance screening silicon nitride single molecule small molecule

项目摘要

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.

描述（由申请人提供）：在纳米孔链测序中，单链 DNA 穿过窄孔，碱基在通过读数头时被识别。在这里，我们重点关注使用α-溶血素（aHL）蛋白纳米孔进行链测序所需的其余任务。纳米孔测序是一种快速实时技术；它不需要耗时的循环添加试剂。在实现具有 106 个孔的芯片后，我们预计到 2014 年，纳米孔测序将在非常短的样品制备时间下实现 15 分钟的基因组测序。此外，纳米孔测序将能够识别修饰碱基并直接对 RNA 进行测序。四年来，我们取得了长足的进步；我们已经证明，所有四种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，并通过纳米孔阵列实现超快速测序。在下一个资助期内，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设计）改进碱基识别，生产适合实时测序的DNA读取头。 2.实现非酶DNA测序的链易位控制。由小分子或工程蛋白环制成的轮烷的使用将减慢DNA运动的速度，以便可以通过现有的记录技术检测碱基。 3. 同时，使用 DNA 聚合酶以酶促方式控制 DNA 运动。该聚合酶还将用于两种新颖的测序模式，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aHL纳米孔的芯片。首先，将开发光学检测106芯片的原型。其次，aHL 孔将被放置在用电子束在氮化硅薄膜上钻孔的孔阵列中，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。