Highly specific, amplification-free, single-molecule counting of rare methylated DNA cancer biomarkers

对罕见甲基化 DNA 癌症生物标志物进行高度特异性、无扩增的单分子计数

• 批准号: 10025913
• 负责人: MUNEESH TEWARI
• 金额: $ 60.85万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-06 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025913
• 关键词: Base Pairing; Benchmarking; Binding; Biological; Biological Assay; Biological Markers; Blood; Blood specimen; Buffers; Cancer Detection; Cancer Diagnostics; Cells; Chemicals; Clinic; Clinical; Colorectal; Colorectal Cancer; Complex; Cytosine; DNA; DNA Damage; DNA Fragmentation; DNA Methylation; DNA Sequence; DNA sequencing; Data; Detection; Diagnostics Research; Discrimination; Dissociation; Early Diagnosis; Epigenetic Process; Equilibrium; Feces; Fingerprint; Fluorescence Microscopy; Genes; Genetic Code; Genomics; Glass; Goals; Gold; Imaging technology; Immobilization; Kinetics; Label; Letters; Malignant Neoplasms; Measures; Methods; Methylation; Microscopy; Modification; Monitor; Mutation; Nucleic Acid Probes; Oligonucleotide Probes; Operative Surgical Procedures; Patients; Plasma; Plasma Cells; Point Mutation; Polymerase Chain Reaction; Protocols documentation; Regulation; Reporting; Research Personnel; Risk; Sampling; Science; Screening for cancer; Sensitivity and Specificity; Series; Site; Slide; Somatic Mutation; Specificity; Surface; Technology; Testing; Uracil; Vision; Work; base; bisulfite; blood-based biomarker; cancer biomarkers; cancer type; cell free DNA; colorectal cancer screening; cost; design; epigenomics; improved; liquid biopsy; methylation biomarker; molecular imaging; mutant; next generation sequencing; novel strategies; patient population; prevent; rare cancer; single molecule; specific biomarkers; success; tumor DNA

PROJECT SUMMARY The ultimate vision of this proposal is to develop a technology platform for the highly specific, rapid, and robust detection of cancer-associated DNA methylation biomarkers for diagnostics and research. Epigenetic alterations are known to be a crucial mode of regulation in cancer. DNA methylation in particular has been known to be dysregulated in cancer for decades and specific loci of DNA methylation 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 methylation at specific sites in DNA. Nearly all established methods require bisulfite treatment of the DNA to convert unmethylated cytosines to uracil (leaving 5-methylcytosine, 5mC, intact), followed by DNA sequencing or quantitative PCR. However, bisulfite treatment is harsh and damages the DNA; obtaining a high efficiency of conversion is typically associated with degradation of >80% of the DNA into poorly amplified fragments. This, when combined with PCR-amplification-based methods, introduces limitations in the sensitivity of detecting methylated DNA at specific sites. Furthermore, the reduced sequence complexity of bisulfite-treated DNA— essentially reducing the four-letter genetic code to a three-letter one—increases the risk of spurious amplification and reduced specificity in quantitative PCR-based approaches. We here propose an approach to DNA methylation 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 fluorescence microscopy to detect the repeated binding and release of sequence-specific fluorescently tagged probes to immobilized target DNA molecules on the surface of a glass slide. Following bisulfite conversion, this unique approach of repetitive probing (i.e., fingerprinting) of single molecules can distinguish between methylated and unmethylated target genes with exquisite specificity (>99.999%) and at the single-molecule level, enabling counting each specific biomarker molecule while avoiding the problem of spurious priming seen with PCR. In contrast to established PCR-based methods, no enzymatic manipulation of the analyte DNA is required with this approach, which is expected to improve sensitivity for highly fragmented input DNA. In order to validate this approach and assess its potential for application to DNA loci important in cancer diagnostics, we propose to develop and benchmark a direct single-molecule DNA methylation assay using kinetic fingerprinting with oligonucleotide probes against loci that are commonly hypermethylated in colorectal cancer, using both synthetic samples and patient blood specimens. By pursuing this work, we will maximize the likelihood of success in developing a transformative new approach for sensitive, specific, PCR amplification-free quantification of cancer-relevant DNA methylation at specific CpG loci.

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甲基化生物标志物的检测。后生 已知改变是癌症中的关键调节模式。特别是DNA甲基化， 几十年来已知在癌症中失调，并且已经寻找了DNA甲基化的特定位点， 血液、粪便和其他样本中癌症的非侵入性生物标志物。然而，进展的一个主要障碍是 缺乏高灵敏度、特异性和定量的方法来检测DNA甲基化， DNA中的特定位点。几乎所有已建立的方法都需要对DNA进行亚硫酸氢盐处理， 将未甲基化的胞嘧啶转化为尿嘧啶（留下5-甲基胞嘧啶，5 mC，完整），然后进行DNA测序或 定量PCR。然而，亚硫酸氢盐处理是苛刻的并且损伤DNA; 转化通常与>80%的DNA降解成扩增差的片段有关。这个， 当与基于PCR扩增的方法结合时， 甲基化DNA在特定位点。此外，亚硫酸氢盐处理的DNA的序列复杂性降低， 从本质上讲，将四个字母的遗传密码减少到三个字母的遗传密码， 在基于定量PCR的方法中，扩增和特异性降低。我们在此提出一种方法， DNA甲基化检测和定量在概念上简单，但利用了复杂的 以及单分子成像科学的优雅进展。该方法是基于使用总内部 反射荧光显微镜检测重复结合和释放的序列特异性 荧光标记的探针与载玻片表面上的固定化靶DNA分子的结合。以下 亚硫酸氢盐转化，这种独特的重复探测方法（即，指纹法）可以 以精确的特异性（>99.999%）区分甲基化和非甲基化靶基因， 单分子水平，能够计数每个特定的生物标志物分子，同时避免 用PCR观察到假引发。与已建立的基于PCR的方法相比，没有酶促操作， 该方法需要分析物DNA，预期其提高高度片段化的灵敏度。 输入DNA为了验证这种方法并评估其应用于DNA位点的潜力， 癌症诊断，我们建议开发和基准的直接单分子DNA甲基化测定 使用寡核苷酸探针的动力学指纹分析， 结直肠癌，使用合成样品和患者血液标本。通过开展这项工作，我们将 最大限度地提高成功开发一种变革性的新方法的可能性， 在特定CpG基因座处癌症相关DNA甲基化的无扩增定量。