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  • Review Article
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Methods for detection of cytosine and thymine modifications in DNA

Nature Reviews Chemistry volume 2, pages 332–348 (2018)Cite this article

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Abstract

Methylation of cytosine at the 5-position is a common epigenetic modification in mammalian DNA and plays an important role in regulating gene expression. Oxidized derivatives of 5-methylcytosine were discovered recently, and some of these oxidized derivatives, in addition to being intermediates in an active demethylation pathway, might also function as epigenetic modifications. Oxidized derivatives of thymine are known to be products of DNA damage, although evidence exists that the oxidized thymine derivative 5-hydroxymethyluracil might have an epigenetic role. There is a pressing need to learn more about these modifications, as epigenetic modifications have roles in development and in diseases, including cancer. This emerging area of research requires highly accurate and sequence-specific methods for the detection of cytosine and thymine modifications in DNA. In this Review, we introduce the biochemistry of cytosine and thymine modifications and discuss established detection methods, such as bisulfite sequencing and its modifications, as well as newer methods, which have been developed to overcome the substantial obstacles associated with studying these modifications in genomic DNA.

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Fig. 1: Active demethylation of cytosine.
Fig. 2: Oxidation of thymine.
Fig. 3: Detection of 5-methylcytosine using N-halogeno-N-sodiobenzenesulfonamide reagents.
Fig. 4: Chemical labelling methods for selective detection of 5-formylcytosine in DNA.
Fig. 5: Oligonucleotide probes for the detection of cytosine modifications.

Adapted with permission from ref.176, RSC.

Fig. 6: Chemical labelling methods for the detection of 5-formyluracil.

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Acknowledgements

The authors’ work was supported by the Irish Research Council (M.B., GOIPG/2017/1453) and the Wellcome Trust Trinity College Dublin (TCD) institutional strategic support fund (J.M.).

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Nature Reviews Chemistry thanks S. Kriaucionis and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Mark Berney & Joanna F. McGouran

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M.B. drafted the initial manuscript. M.B. and J.M. contributed equally to the discussion of the content and the reviewing and editing of the article.

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Glossary

Transcription factors

Proteins that bind to specific DNA sequences and thereby regulate the transcription of genes.

Abasic site

A site in DNA that consists of a deoxyribose unit lacking a purine or pyrimidine base.

Base excision repair

(BER). A cellular DNA repair pathway that removes and replaces damaged or mismatched nucleobases in DNA.

Promoters

Regions of DNA close to transcription start sites, which control transcription initiation by providing binding sites for transcription factors and RNA polymerase.

Poised and active enhancers

DNA sequences to which transcription factors bind to regulate the transcription of associated genes.

Exons

Sequences within genes that encode the amino acids in proteins. Unlike introns, exons are not removed from the RNA transcript during splicing.

Affinity isolation

The use of non-covalent interactions with a protein to facilitate the isolation of a target molecule.

Restriction endonucleases

Enzymes found in bacteria and archaea that are used for antiviral defence and that cleave DNA at or near specific recognition sites.

Electrogenerated chemiluminescence

A process in which reactive intermediates that are generated electrochemically undergo reactions to form excited-state species that emit light.

Excimer

A dimer formed from two monomers, one of which is in an excited electronic state.

Locked nucleic acid

(LNA). A synthetic oligonucleotide analogue in which ribose units are conformationally restricted by a 2ʹ-O-4ʹ-C methylene bridge.

Peptide nucleic acid

A synthetic oligonucleotide analogue in which the sugar–phosphate backbone is replaced by a peptide chain.

Duplex melting temperature

The temperature at which half the DNA strands in a solution are found in a single-stranded state, which is used as a measure of duplex stability.

Fluorescence in situ hybridization

(FISH). A technique for detecting specific sequences in chromosomal DNA using a fluorescent oligonucleotide probe that binds to a target site on a chromosome and is then visualized by fluorescence microscopy.

Droplet digital PCR

A procedure in which template DNA molecules are partitioned into droplets in a water–oil emulsion. PCR amplification occurs within each droplet and is then analysed to determine the fraction of droplets in which the PCR was successful, enabling the concentration of template DNA in the original sample to be determined.

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Berney, M., McGouran, J.F. Methods for detection of cytosine and thymine modifications in DNA. Nat Rev Chem 2, 332–348 (2018). https://doi.org/10.1038/s41570-018-0044-4

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