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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结