The hunt for 5-hydroxymethylcytosine: the sixth base.

transfers a glucose from UDP-glucose to 5-hmC in T4 bacteriophages. Two groups independently reported the use of b-GT to detect and quantify 5-hmC. Szwagierczak et al. used H radioactive glucose to label and quantify 5-hmC by scintillation counting [10]. Song et al. used b-GT to transfer an azide-labeled glucose to 5-hmC and subsequently attached a biotin group through the azido group via copper-free click reaction [11]. This strategy allows for the detection and quantification of 5-hmC in a simple dot-blot assay using avidin-horseradish peroxidase. The approach of b-GT labeling greatly improved the detection limit of 5-hmC from 0.004–0.006% of total nucleotide, and displayed a linear response to 5-hmC since the b-GT-based method is able to label every single 5-hmC in the genome. With these highly sensitive methods, extremely low levels of 5-hmC in other cultured cells, especially cancer cells, can be measured [11]. This is of particular interest since Tet enzymes may play important roles in various cancers; however, cancer cell lines tend to have low levels of 5-hmC [11,12]. The b-GTbased labeling approaches represent promising diagnostic tools for specific cancers. Compared to simple 5-hmC detection, sequencing methods amenable for wholegenome, next-generation sequencing of 5-hmC are much more desirable, especially if 5-hmC-containing genomic DNA fragments can be enriched first. We generated the first genome-wide 5-hmC distribution map using the same b-GT/azide–glucose/biotin-labeling method [11]. The high specificity of the click chemistry and the highly selective and strong biotin-avidin/streptavidin interaction enabled the robust and efficient pull-down of 5-hmCcontaining DNA for deep sequencing. The biotin–avidin/streptavidin enrichment approach proved superior to the antibody-based pull down After its rediscovery in 2009, 5-hydroxymethylcytosine (5-hmC) and its natural creators, the Tet dioxygenases, have received a tremendous amount of attention from the epigenetics and other related communities [1,2]. 5-hmC is now widely accepted as the sixth base in the mammalian genome, following its precursor, 5-methylcytosine (5-mC), the fifth base. To elucidate the exact biological roles of this base modification, the development of new analytical technologies, including detection and sequencing methods for 5-hmC, has become essential. The f irst two reports in 2009 used P-radioactive labeling 1D or 2D thin layer chromatography to detect 5-hmC [1,2]. With a detection limit of approximately 0.08% of total nucleotide, the thin layer chromatography method is able to detect 5-hmC in genomic DNA from brain tissue and embryonic stem cells; however, it fails to yield a signal with genomic DNA isolated from other cultured cells, especially cancer cells. More recently, a more accurate HPLC-mass spectroscopy method has been developed with isotope-labeled 5-hmC as an internal standard [3]. This method allows a complete quantification of the 5-hmC in all mouse tissues; however, the detection limit still needed to be improved. Since then anti-5hmC antibodies have become available and are widely used to detect 5-hmC [4–7]. However, this approach is not very quantitative owing to its density-dependent recognition of 5-hmC, as expected [8]. Anti-5-methylenesulfonate – generated from 5-hmC after a bisulfite treatment – antibodies have been demonstrated to be more quantitative than anti-5-hmC antibodies [8]. However, these antibody-based methods c annot improve the detection limit. In 2010, enzymatic labeling of 5-hmC began to emerge [9]. T4-b-glucosyltransferase (b-GT) is the first enzyme of choice, since it naturally