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

> **NIH NIH R21** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2020 · $608,541

## Abstract

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...

## Key facts

- **NIH application ID:** 10025913
- **Project number:** 1R21CA225493-01A1
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** MUNEESH TEWARI
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $608,541
- **Award type:** 1
- **Project period:** 2020-08-06 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10025913

## Citation

> US National Institutes of Health, RePORTER application 10025913, Highly specific, amplification-free, single-molecule counting of rare methylated DNA cancer biomarkers (1R21CA225493-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10025913. Licensed CC0.

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