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There is substantial interest and speculation in the role of the "sixth DNA base", 5-hydroxymethylcytosine (5-hmC), although its precise function has not yet been elucidated. This recently " rediscovered " cytosine base modification results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases (Kriaucionis and Heintz, 2009 ; Tahiliani et al., 2009 ; Loenarz and Schofield, 2009 ; Iyer et al. 2009). Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics: 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting hmC to C and, as such open up entirely new perspectives in epigenetic studies (Ito et al., 2010). Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (eg. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC (Huang et al., 2010 ; Jin et al, 2010 ; Nestor et al., 2010). Another factor that makes hmC difficult to study is its relatively low abundance. In ES cells, hmC is ~4% of all cytosine species in CpG dinucleotides located in MspI cleavage sites (CCGG). CpG is ~0.8% of all dinucleotides in the mouse genome, thus hmC constitutes ~0.032% of all bases or ~1 in every 3000 nucleotides. For comparison, 5mC is 55-60% of all cytosines in CpG dinucleotides in MspI cleavage sites, about 14-fold higher than hmC. Therefore a full reappreciation of the biological significance of hmC will depend heavily on the tools and methods that allow hmC, 5mC and C to be distinguished unequivocally and effciently. The development of the affinity-based technologies appears to be the most powerful way and so far the only way to differentially and specifically enrich 5mC and 5hmC sequences from complex sources such as genomic DNA.
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