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为高度特定，快速且健壮的高度开发技术平台 检测与癌症相关的DNA甲基化生物标志物进行诊断和研究。表观遗传学 已知改变是癌症调节的至关重要方式。尤其是DNA甲基化已经 已知在癌症中失调数十年，并且已经厌含着DNA甲基化的特定位置 血液，凳子和其他样品中癌症的非侵入性生物标志物。但是，进步的主要障碍 缺乏用于检测DNA甲基化的高度敏感，特异性和定量方法 DNA中的特定位点。几乎所有已建立的方法都需要Bisulfite治疗DNA才能转换 未甲基化的胞嘧啶至尿嘧啶（留下5-甲基胞嘧啶，5MC，完整），然后进行DNA测序或 定量PCR。但是，硫酸盐治疗是危害的，并损害了DNA。获得高效率 转化通常与> 80％的DNA的定义相关联，分为不良的片段。这， 当与基于PCR放大的方法结合使用时，引入了检测灵敏度的局限性 在特定部位的甲基化DNA。此外，Bisulfite处理的DNA的序列复杂性降低 从本质上，将四个字母的遗传密码降低到三个字母的遗传密码 - 伪造的风险 基于定量PCR的方法的扩增和降低的特异性。我们在这里提出了一种方法 DNA甲基化检测和定量在概念上很简单，却充分利用了复杂的优势 和单分子成像科学的优雅进步。该方法基于使用总内部 反射荧光显微镜，以检测序列特异性的重复结合和释放 荧光标记为固定的靶DNA分子在载玻片表面上。下列的 亚硫酸氢盐转化，这种独特的重复探测（即指纹）单分子的方法可以 具有独特特异性（> 99.999％）的甲基化和未甲基化靶基因之间的区别 单分子水平，可以计算每个特定的生物标志物分子，同时避免 使用PCR看到的虚假启动。与已建立的基于PCR的方法相反，没有酶促操作 这种方法需要分析物DNA，预计将提高对高度碎片的敏感性 输入DNA。为了验证这种方法并评估其应用于DNA基因座的潜力 癌症诊断，我们建议开发和基准直接的单分子DNA甲基化测定法 使用动力学指纹与寡核苷酸问题对基因座的寡核苷酸问题，通常是高甲基化的 结直肠癌，同时使用合成样品和患者血液标本。通过从事这项工作，我们将 最大程度地提高成功开发敏感，特定，PCR的新方法的可能性 在特定CpG基因座上对癌症相关的DNA甲基化的无扩增定量。