hMeDIP-chip performed on human brain DNA using 5-Hydroxymethylcytosine (5-hmC) antibody. Human brain DNA (2 ug) was immunoprecipitated with 10 ug of 5-Hydroxymethylcytosine antibody. Following hMeDIP, the DNA was amplified, labeled and hybridized to an Affymetrix Human Tiling 2.0R Array. Shown is a region from chromosome 6q containing the ARID1B, ZDHHC14 and SNX9 genes. The results show that 5-hydroxymethylcytosine is enriched primarily in the coding regions of genes, rather than in the gene promoter or regulatory regions.
5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by Methyl DNA immunoprecipitation. DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 2 μg of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 2 μg of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC).
5-Hydroxymethylcytosine (5-hmC) antibody (mAb) tested by dot blot analysis. DNA samples were spotted (indicated in ng on the left) on to a positively charged nylon membrane and blotted with 5-Hydroxymethylcytidine antibody at a 0.2 mg/ml dilution. Lane 1: double-stranded DNA containing 5-hydroxymethylcytosine. Lane 2: double-stranded DNA containing 5-methylcytosine. Lane 3: unmethylated double-stranded DNA. Lane 4: single-stranded DNA containing 5-hydroxymethylcytosine. Lane 5: single-stranded DNA containing 5-methylcytosine. Lane 6: unmethylated single-stranded DNA.
Dot blot of 5-Hydroxymethylcytosine (5-hmC) mAb. Dot blot analysis was used to confirm the specificity of 5-Hydroxymethylcytosine antibody for 5-hydroxymethylcytidine. 10 ng of single-stranded 38 nt DNA oligonucleotides were spotted onto nitrocellulose and probed with the antibody at 0.2mg/mL. Lane 1: oligo containing unmodified cytidine. Lane 2: oligo containing 5-methylcytidine. Lane 3: oligo containing 5-hydroxymethylcytidine. Lane 4: oligo containing 5-formylcytidine. Lane 5: oligo containing 5-carboxylcytidine.
