Methods of detecting additional epigenetic modifications

This 2018 German review subject was detecting DNA modifications that are derivatives of the much-studied 5-methlycytosine:

“The discovery of modified nucleobases arising from 5-methylcytosine (5mC) through consecutive oxidation to give 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) has stimulated intense research efforts regarding the biological functions of these epigenetic marks.

Recent findings revealed that 5hmC and 5fC are stable DNA modifications in the genome, thus suggesting that oxidized 5mC derivatives may function as epigenetic marks in their own right, exhibiting regulatory purposes and participating in DNA replication, transcription, repair, and recombination.

The bisulfite-sequencing method (BS-Seq) has widely been used as the gold standard in determining the methylation status with single-base resolution in genomic DNA. The BS-Seq method, however, has some severe drawbacks, such as:

  • Harsh reaction conditions which might cause undesired DNA damage,
  • Requirements for relatively large amounts of input DNA,
  • Dependence on PCR, and resulting short sequence reads, as well as
  • Reduced sequence complexity due to deamination of all nonmethylated cytosines and
  • Accompanied challenges for primer hybridization.

Most importantly, however, with BS-Seq it is not possible to discriminate between 5mC and 5hmC..Furthermore, since 5fC and 5aC undergo deamination similar to unmodified cytosine, they are indistinguishable from C under bisulfite conditions.” “Chemoselective labeling and site-specific mapping of 5-formylcytosine as a cellular nucleic acid modification” (click the PDF link)

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