Molecular Reading Head for Single-Molecule DNA sequencin

单分子 DNA 测序分子读头

• 批准号: 7058801
• 负责人: STUART LINDSAY
• 金额: $ 18.25万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-14 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7058801
• 关键词: atomic force microscopy; cyclodextrins; genome; glucans; hydrocarbons; molecular probes; nanotechnology; nucleic acid sequence; polypropylenes; protein structure; small molecule; technology /technique development; thermodynamics

DESCRIPTION (provided by applicant): The goal of the proposed study is to evaluate a novel single-molecule DNA sequencing technology that has the potential to sequence a molecule of genomic dimension in hours. The DNA is attached to a rotaxane complex consisting of a molecular ring (cyclodextrin) that self-threads onto a propylene oligomer. The far end of the propylene oligomer is attached to a fixed surface, and the cyclodextrin ring is covalently attached to an AFM probe. As the AFM probe is pulled away from the fixed surface, the DNA passes through the cyclodextrin ring, one base at a time. Fluctuations in molecular friction as the ring passes each base are recorded as deflections of the AFM cantilever. If these data can be interpreted in terms of the base sequence of long DNA molecules, then single DNA molecules can be sequenced rapidly with this new technology. Preliminary studies appear to show that the DNA can be pulled through a cyclodextrin ring. They also indicate that the cantilever deflection during retraction depends on the DNA sequence. An unanticipated discovery is that double stranded DNA appears to pass the ring more easily than single stranded DNA, and does so with less random fluctuation than is the case for single stranded DNA. Our first goal is to put the 'ring sliding' model to further test. Does the ring really slide over one strand of double-stranded DNA, peeling the complementary strand off? Does sequence-specifc adhesion between the DNA and the fixed surface contribute to the sequence-related signal? If these experiments unearth a problem with our system, we will modify the chemistry appropriately. Once the operation of the system is verified, we will carry out a program of theory and experiment aimed at understanding these initial observations and establishing the limitations of the technology as developed thus far. Guided with information from these studies, improved molecular 'reading heads' will be designed, sequencing parameters will be optimized and hardware will be improved with the goal of reliable sequencing of oligomers, a pre-requisite for subsequent attempts at large-scale sequencing.

描述（由申请人提供）：拟议研究的目的是评估一种新型的单分子DNA测序技术，该技术有可能在小时内对基因组维度分子进行测序。 DNA连接到由由分子环（环糊精）组成的轮济烷络合物，该复合物自动读取在丙烯低聚物上。 丙烯低聚物的远端附着在固定表面上，并且环糊精环共同附着在AFM探针上。 当将AFM探针从固定表面拉开时，DNA一次穿过一个碱基。 当环通过时，分子摩擦的波动记录为AFM悬臂的偏转。 如果可以根据长DNA分子的基础序列来解释这些数据，则可以用这种新技术快速对单个DNA分子进行测序。 初步研究似乎表明DNA可以通过环糊精环拉出。 它们还表明，缩回过程中的悬臂挠度取决于DNA序列。 一个意外的发现是，双链DNA似乎比单链DNA更容易地通过环，并且与单链DNA相比，随机波动较少。 我们的第一个目标是将“环形滑动”模型放置以进一步测试。 环是否真的滑过一条双链DNA，将互补的链剥离？ DNA和固定表面之间的序列特异性粘附是否有助于序列相关信号？ 如果这些实验发现我们的系统问题，我们将适当修改化学。 一旦验证了系统的运行，我们将执行一个理论和实验计划，旨在理解这些最初的观察，并确定到目前为止开发的技术的局限性。 通过这些研究的信息进行指导，将设计改进的分子“阅读头”，将对测序参数进行优化，并以可靠的低聚物测序的目的改进硬件，这是对随后尝试大规模测序的前提。

项目成果

An AFM/rotaxane molecular reading head for sequence-dependent DNA structures.

• DOI: 10.1002/smll.200800233
• 发表时间: 2008-09
• 期刊: SMALL
• 影响因子: 13.3
• 作者: Ashcroft, Brian A.;Spadola, Quinn;Qamar, Shahid;Zhang, Peiming;Kada, Gerald;Bension, Rouvain;Lindsay, Stuart
• 通讯作者: Lindsay, Stuart

Can an atomic force microscope sequence DNA using a nanopore?

• DOI: 10.1529/biophysj.107.108670
• 发表时间: 2008-02-15
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Qamar, Shahid;Williams, Phil M.;Lindsay, S. M.
• 通讯作者: Lindsay, S. M.