Cytosine, one of the four DNA bases, can be chemically modified by the addition of a molecule known as a methyl group to form 5-methylcytosine. This “epigenetic” modification has long been known to regulate gene expression and plays a critical role in processes like embryonic development. Its levels and distribution are also distinct in different tissues and are significantly altered in cancers. Analysing methylation patterns of DNA shed into blood and other bodily fluids by tumours can thus reveal both the presence and the location of a cancerous growth.
In 2019, Dr Chunxiao Song (Ludwig Institute for Cancer Research, Oxford Branch, Nuffield Department of Medicine) and his team developed TET-assisted pyridine borane sequencing (TAPS) for mapping DNA methylation. The technology was spun out in 2020 to establish the biotechnology company Base Genomics, which was acquired for $410 million by Exact Sciences in October 2020. Compared to the previous gold standard for sequencing DNA methylation, TAPS is far more cost-effective and sensitive, and generates cleaner data to allow for additional genetic analysis.
Yet despite its advantages, TAPS still relies on whole-genome sequencing, which remains an expensive approach for detecting DNA methylation since just ~4% of all cytosines in the genome are methylated. Chunxiao and his team have now developed a new method that cuts costs further by sequencing only those regions of the genome that contain methylated cytosines.
Building on the TAPS method, postdocs Dr Jingfei Cheng and Dr Paulina Siejka-Zielińska made use of molecular scissors called endonucleases that recognise and cut specific DNA sites. During TAPS, methylated cytosines are chemically converted to an altered base called dihydrouracil (DHU). The researchers found an endonuclease called USER enzyme that specifically cuts at DHU. Because of the enzyme specificity, they knew that all the DNA fragments produced had methylation sites at the beginnings and ends. By then size-selecting the DNA to exclude the larger, uncut DNA, only the smaller, cut DNA fragments with methylation sites are sequenced, making this approach more cost-effective for studying DNA methylation at base-pair resolution.
The team has named the new technique endonuclease enrichment TAPS (eeTAPS), and details on the method can be found in their publication in Nucleic Acids Research.