A new report presents DM-Seq (direct methylation sequencing), a bisulfite-free method for profiling 5-methylcytosine (5mC) at single-base resolution using nanogram quantities of DNA. The technique allows scientists to profile DNA using very small samples and without damaging the sample, making it a potential candidate for use in liquid biopsies and early cancer detection. Additionally, unlike current methods, it also can clearly identify 5mC without confusing it with other common markings.

This work is published in Nature Chemical Biology in the paper, “Direct enzymatic sequencing of 5-methylcytosine at single-base resolution.

“5mC can act as a fingerprint for cell identity, so it’s important for scientists to have the power to isolate 5mC and only 5mC,” said Rahul Kohli, MD, PhD, an associate professor of biochemistry and biophysics at the University of Pennsylvania, School of Medicine. “DM-Seq uses two enzymes to map 5mC and can be applied to sparse DNA samples which means it could be used, for example, in blood tests that look for DNA released into the blood from tumors or other diseases tissues.”

The abnormal absence or excess of 5mC can lead to abnormal gene expression, which can drive diseases such as cancers. In fact, certain abnormal patterns of 5mCs are considered signatures of some cancers—which underscores the importance of having an accurate and specific 5mC mapping method.

The traditional method for mapping 5mC, bisulfite sequencing (BS-Seq), is significantly damaging to DNA, and fails to distinguish between 5mC and other types of methylation such as 5-hydroxymethylcytosine (5hmC). More recently developed methods also have shortcomings including the requirement for relatively large amounts of DNA.

DM-Seq utilizes two enzymes, a designer DNA methyltransferase and a DNA deaminase, which together can detect 5mC directly and specifically. Coupling these activities with deaminase-resistant adapters, noted the authors, enables accurate detection of only 5mC via a C-to-T transition in sequencing.

The researchers performed DM-Seq on glioblastoma-type brain tumor samples and demonstrated that, in comparison with traditional BS-Seq, DM-Seq was better able to distinguish 5mC from 5hmC at key sites on the genome where methylation levels can be used to predict patient outcomes.

The researchers also compared DM-Seq to another new, emerging 5mC-sequencing technique known as TET-assisted pyridine borane sequencing (TAPS), which is being explored for potential applications in cancer diagnostics, showing that the latter has a previously undiscovered drawback that reduces its 5mC-detection sensitivity.

“These findings highlight ways in which direct detection of 5mC from DM-Seq, rather than traditional sequencing methods, could advance efforts to use epigenetic sequencing for prognostic purposes in cancer care,” Kohli said.

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