Droplet Digital™ PCR technology unlocks the diagnostic power of DNA methylation

Explore the benefits of a combined methylation-sensitive restriction enzyme and ddPCR approach to detect DNA methylation in healthy and disease states.

4 Jul 2025

DNA methylation is emerging as a key biomarker for diagnosis of cancer and other diseases. As such, there is an increased interest in non-invasive, sensitive tools for detecting disease early. Eddy van Collenburg, Market Development Specialist at Bio-Rad Laboratories, shares how ddPCR, combined with methylation-sensitive restriction enzymes (MSREs) offers a powerful and precise alternative to traditional methylation detection methods.

Eddy van Collenburg, Bio-Rad

We’ve always been searching for the perfect marker, and methylation might be the closest thing we have. It's a pan-disease, even pan-cancer, biomarker with tremendous potential for diagnosis and monitoring.

Eddy van Collenburg  Market Development Specialist, Bio-Rad

Improving on the gold standard of DNA methylation detection

DNA methylation, a key epigenetic mechanism within cells involving the addition of a methyl group to cytosine bases, plays a critical role in gene regulation, cellular identity, and genomic stability. Alterations in methylation patterns are increasingly recognized as early indicators of pathological changes in cancer, neurological disorders, cardiovascular disease, and aging. Methylation is gaining recognition as a powerful biomarker with wide-ranging diagnostic and prognostic potential.

However, detecting subtle changes in methylation status (or profile), especially at early disease stages, remains a significant technical challenge. Current gold standard methods – such as bisulfite sequencing – are labor intensive and can result in moderate to extensive damage to DNA. “Bisulfite sequencing involves chemically treating DNA to convert unmethylated cytosines to uracil,” van Collenburg explained. “But this process causes substantial sample loss, often 50% or more, and is particularly problematic when working with typically limited sample inputs from liquid biopsies. The treatment is harsh on DNA and adds complexity to the workflow.”

To overcome these limitations, Bio-Rad has developed an alternative approach using MSREs in combination with ddPCR. The method avoids chemical conversion and the associated degradation potential whilst maintaining high levels of analytical sensitivity.

“We’ve combined MSREs with ddPCR to create an assay that’s both robust and gentle on limited sample inputs,” comments van Collenburg. “The combination of MSRE to detect methylation status at a particular locus followed by detection using ddPCR has demonstrated that methylation status can be determined to a high level of sensitivity, with inputs as low as 1 ng. Using this method, we have already successfully designed and tested over 50 of the most published oncology methylation targets.”

In this method, MSREs selectively digest unmethylated DNA at specific recognition sites, while methylated regions remain protected. The cleaved and uncleaved DNA is then quantified using ddPCR. Since the assay relies on restriction enzyme activity, amplicon design must incorporate cleavage sites. To increase flexibility and specificity, a wide range of MSREs and cut sites are available for customized combinations.

Applications of the MSRE ddPCR approach to DNA methylation detection

From oncology to organ transplants, the applications and impact of DNA methylation detection are broad. The long-term goals of the Bio-Rad team are that MSREs will be used not only for early cancer detection and monitoring but also for other translation research areas such as transplant rejection, cardiovascular incidents, neurological changes, and aging. “We’re seeing methylation analysis being used to monitor organ transplant rejection, undetected heart attacks, and neurological degeneration. It’s far more than just oncology now,” van Collenburg noted.

There is hope that proactive methylation-based screening could be used to help identify disease risks before symptoms occur, to enable more effective early interventions. “Modern medicine is shifting from treatment to prevention, and methylation could be the tool that makes this transformation possible,” shares van Collenburg.

The shift to an assay format that utilises a small volume liquid biopsy, with no harsh chemical treatments, is critical to the ultimate goal of a rapid patient test in a preventative or monitoring scenario.

As the field continues to evolve, further advances in assay design, automation, and regulatory standardization will be paramount in scaling use in the clinic. Still, the promise is clear: combining MSREs with ddPCR ensures that samples can be analyzed with high sensitivity, specificity, and practicality.

Learn how to integrate methylation detection into your workflows with Bio-Rad’s ddPCR methylation assays, and download Bio-Rad’s latest application note for free from SelectScience to get started today.

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