Related Experiment Video
Updated: May 28, 2026
08:23
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Establishment and Performance Evaluation of a Multiplexed TET2-APOBEC-Mediated cfDNA Methylation Detection Workflow
Almudena Aguilera-Diaz1, Philip B Feinberg1, Jianmin Huang1
1Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY 10065, USA.
Journal of Personalized Medicine
|May 26, 2026
Summary
A new enzymatic assay for cell-free DNA (cfDNA) methylation detection offers sensitive, quantitative results without DNA degradation. This method supports early colorectal cancer screening and liquid biopsy applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Bisulfite-based cell-free DNA (cfDNA) methylation assays are valuable for biomarker detection.
- These assays often degrade DNA and have inconsistent conversion efficiency, limiting their use in liquid biopsies.
Purpose of the Study:
- To develop and evaluate a fully enzymatic cfDNA methylation workflow.
- The goal was to preserve DNA integrity and enable quantitative clinical detection.
Main Methods:
- Integrated TET2-mediated oxidation and APOBEC3A deamination.
- Utilized RNase H2-guided primer design, uracil-DNA glycosylase error suppression, and dual-probe detection.
- Assessed performance using serial dilutions and plasma cfDNA from colorectal cancer (CRC) patients and healthy donors.
Main Results:
- The 40-marker panel showed higher methylation scores in CRC cfDNA.
- Digital PCR (dPCR) demonstrated single-molecule resolution and sensitivity below 0.2% methylated DNA.
- Quantitative PCR (qPCR) and dPCR showed strong correlation (R² = 0.80) and high concordance in sample classification.
Conclusions:
- The TET2-APOBEC-based enzymatic cfDNA assay provides sensitive, quantitative, sequencing-free methylation detection.
- The gentle conditions preserve DNA integrity, suitable for low-input samples.
- The assay supports early colorectal cancer screening and clinical liquid biopsy workflows.
