Molecular Reading Head for Single-Molecule DNA sequencin

单分子 DNA 测序分子读头

• 批准号: 6894078
• 负责人: STUART LINDSAY
• 金额: $ 18.69万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6894078
• 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(申请人提供)：拟议研究的目标是评估一种新的单分子DNA测序技术，该技术具有在数小时内对基因组尺寸的分子进行测序的潜力。DNA被连接到轮烷复合体上，该复合体由一个分子环(环糊精)组成，该分子环自穿接到丙烯齐聚物上。丙烯齐聚物的远端固定在固定的表面上，环糊精环共价连接在AFM探针上。当AFM探针离开固定表面时，DNA通过环糊精环，一次一个碱基。当环经过每个基座时，分子摩擦的波动被记录为AFM悬臂的偏转。如果这些数据可以用长DNA分子的碱基序列来解释，那么单个DNA分子就可以用这项新技术快速测序。 初步研究似乎表明，DNA可以通过环糊精环拉出。它们还表明，回缩过程中的悬臂偏转取决于DNA序列。一个意想不到的发现是，双链DNA似乎比单链DNA更容易通过环，而且这样做的随机波动比单链DNA的情况要小。 我们的第一个目标是对“环滑动”模型进行进一步测试。这个环真的会滑过一条双链DNA，把互补的DNA链剥离吗？DNA和固定表面之间的序列特异性黏附对序列相关信号有贡献吗？如果这些实验发现我们的系统有问题，我们将适当地修改化学成分。 一旦系统的运行得到验证，我们将进行一项理论和实验计划，旨在了解这些初步观察结果，并确定迄今开发的技术的局限性。在这些研究的指导下，将设计改进的分子“读数头”，优化测序参数，改进硬件，以实现低聚物可靠测序的目标，这是随后尝试大规模测序的先决条件。