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

项目总结 这项提议的最终愿景是为直接、快速和强大的 用于诊断和研究的癌症相关DNA突变检测。DNA突变是已知的 几十年来一直是癌症发展的基础，突变的DNA的特定片段 寻求作为血液、粪便和其他样本中癌症的非侵入性生物标志物。然而，一个主要的障碍是 进展是缺乏检测dna突变的高度敏感、特异和定量的方法。 当突变的DNA片段作为稀有等位基因出现在高背景的野生型DNA中时。我们建议 这里是一种DNA突变检测和量化的方法，从概念上来说很简单，但需要 单分子成像科学的尖端和优雅进步的优势。这种方法是基于 用全内反射显微镜检测荧光标记探针的结合和释放 将目标DNA分子固定在玻片表面。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，这影响了 以单分子显微镜可以检测到的方式结合和释放探针。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 执行可提高敏感度和吞吐量的优化，并使用 来自肺癌患者和对照的临床样本。

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.