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Optimization and Validation of Single-Molecule Kinetic Fingerprinting Technology for Rapid, Ultra-Specific Detection of Cancer Mutations

用于快速、超特异性检测癌症突变的单分子动力学指纹技术的优化和验证

基本信息

批准号：
10000971
负责人：
MUNEESH TEWARI
金额：
$ 38.84万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-13 至 2021-08-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY The ultimate vision of this proposal is to develop a technology platform for the direct, rapid, and robust detection of cancer-associated DNA mutations for diagnostics and research. DNA mutations have been known to be fundamental to cancer development for decades and specific fragments of mutated DNA have been sought as non-invasive biomarkers for cancer in blood, stool and other samples. However, a major barrier to progress has been the lack of highly sensitive, specific, and quantitative methods for detecting DNA mutation when mutant DNA fragments are present as rare alleles in a high background of wild-type DNA. We propose here an approach to DNA mutation detection and quantification that is conceptually simple, yet takes advantage of sophisticated and elegant advances in single molecule imaging science. The approach is based on using total internal reflection microscopy to detect the binding and release of a fluorescently-tagged probe to immobilized target DNA molecules on the surface of a glass slide. Differences in nucleotide sequence even at a single nucleotide, give rise to differences in the free energy of hybridization, and this affects the kinetics of binding and release of a probe in a manner that can be detected by single-molecule microscopy. We have already performed proof-of-concept and established feasibility of the approach. In this project, we aim to perform optimizations that will increase sensitivity and increase throughput, and to perform validation with clinical samples from lung cancer patients and controls.

项目摘要该提案的最终愿景是开发一个技术平台，用于诊断和研究的癌症相关DNA突变的检测。已知DNA突变几十年来，突变DNA的特定片段一直是癌症发展的基础，在血液、粪便和其他样品中寻求作为癌症的非侵入性生物标志物。然而，目前的进展是缺乏高灵敏度、特异性和定量的DNA突变检测方法当突变DNA片段作为稀有等位基因存在于野生型DNA的高背景中时。我们提出这里，一种概念上简单但需要单分子成像科学的复杂和优雅的进步的优势。该方法是基于使用全内反射显微镜检测荧光标记探针的结合和释放固定在载玻片表面的靶DNA分子。核苷酸序列的差异甚至在单个核苷酸上，引起杂交自由能的差异，这会影响杂交的动力学。探针的结合和释放可以通过单分子显微镜检测到。我们有已经进行了概念验证，并确定了该方法的可行性。在这个项目中，我们的目标是执行优化，以提高灵敏度和吞吐量，并执行验证来自肺癌患者和对照的临床样品。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning.

DOI：
10.1038/s41467-020-19673-1
发表时间：
2020-11-17
期刊：
Nature communications
影响因子：
16.6
作者：
Li J;Zhang L;Johnson-Buck A;Walter NG
通讯作者：
Walter NG

A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

DOI：
10.1016/j.ymeth.2021.06.014
发表时间：
2022-01
期刊：
Methods (San Diego, Calif.)
影响因子：
0
作者：
Mandal S;Khanna K;Johnson-Buck A;Walter NG
通讯作者：
Walter NG

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

DOI：
10.1021/acs.nanolett.2c01757
发表时间：
2022-08-10
期刊：
NANO LETTERS
影响因子：
10.8
作者：
Blanchard, Aaron T.;Li, Zi;Duran, Elizabeth C.;Scull, Catherine E.;Hoff, J. Damon;Wright, Keenan R.;Pan, Victor;Walter, Nils G.
通讯作者：
Walter, Nils G.

Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.

DOI：
10.1016/j.bios.2021.113433
发表时间：
2021-10-15
期刊：
Biosensors & bioelectronics
影响因子：
12.6
作者：
Khanna K;Mandal S;Blanchard AT;Tewari M;Johnson-Buck A;Walter NG
通讯作者：
Walter NG

Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.

DOI：
10.1021/acs.accounts.0c00621
发表时间：
2021-01-19
期刊：
Accounts of chemical research
影响因子：
18.3
作者：
Mandal S;Li Z;Chatterjee T;Khanna K;Montoya K;Dai L;Petersen C;Li L;Tewari M;Johnson-Buck A;Walter NG
通讯作者：
Walter NG