A new method of single-molecule detection of 5-hydroxymethylcytosine (5hmC) in DNA has been developed.
Selective thiol substitution of 5hmC (giving SMC) in a single-step, bisulfite-mediated reaction allows the incorporation of a peptide (yellow sphere) or biotin into DNA. Modified 5hmC bases can be readily distinguished at the single-molecule level using protein nanopore analysis.
To date, the identification of hydroxymethylcytosine has required complex, expensive, or error-prone processes. A team led by Hagan Bayley at the University of Oxford has now developed a chemical modification that allows for the differentiation of hydroxymethylcytosine and methylcytosine through sequencing in nanopores.
Developed by Oxford Nanopore, a company formed by Hagan Bayley in 2005, the nanopore method is a highly promising alternative to the sequencing of individual DNA molecules without an amplification step. Fed by an enzyme, a single strand of DNA threads through a membrane-embedded protein pore. Depending on which of the bases is in the narrowest part of the pore at a given time, there is a characteristic change in the flow of current through the pore.
A chemical reaction between hydroxymethylcytosine, bisulfite, and a cysteine-containing peptide that leaves the other bases—including methylcytosine—unchanged, greatly improves the resolution as the various bases result in differences in current.
Importantly, it is possible to attach a fluorescent marker to the modified site, or a molecular “eye” that can be used to attach the rare hydroxymethylcytosine-containing DNA fragments to “hooks” that allow the fragments to be enriched over unmodified fragments, enabling rapid sequence analysis.
Li, Gong and Bayley, “Single-Molecule Detection of 5-Hydroxymethylcytosine in DNA through Chemical Modification and Nanopore Analysis”. Angewandte Chemie, Vol. 52, Issue 16, pp. 4350–4355 (2013)