Chemical Methods for Decoding Cytosine Modifications in DNA

The decoding of DNA falls within the general scope of chemical structure elucidation and has been naturally enabled by the creation and application of chemical approaches. Decoding the sequence of the four canonical DNA bases was first made widely accessible in the late 1970s with two independent chemical approaches from Maxam and Gilbert and from Sanger. It is now possible to decode 5mC, 5hmC, 5fC, and 5caC in addition to G, C, A, and T in DNA at single base resolution. One approach for the single molecule sequencing of modified bases is to exploit the pausing of a polymerase due to the presence of chemical tags; this has been demonstrated for the detection of 5hmC in single-molecule real-time sequencing (SMRT). SMRT DNA sequencing is a single molecule sequencing technology, whereby the continuous incorporation of phospholinked nucleotides by a DNA polymerase is detected as fluorescent pulses. This method represents the first example of a single molecule sequencing method being employed to detect 5hmC at single base resolution.