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5-hmC/5-mC DNA Labeling

An easy and cost-efficient method for the preparation of 5-(hydroxy)methylated DNA fragments relies on 5-hydroxmethyl-dCTP (5-hmdCTP) or 5-methyl-dCTP (5-mdCTP) that is enzymatically incorporated into DNA with Taq polymerase instead of its natural counterpart dCTP.

5-(hydrox)ymethylated DNA fragments can subsequently be used as sequencing control [1-5] or for pull-down of 5-hydroxymethyl-binding proteins from cellular lysate[6].

Selected references

[1] Booth et al. (2014) Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat. Chem. 6 (5):435.
[2] Booth et al. (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8 (10):1841.
[3] Yu et al. (2012) Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat. Protoc. 7 (12):2159.
[4] Szwagierczak et al. (2011) Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine. Nucl. Acids Res. 39(12):5149.
[5] Szwagierczak et al. (2010) Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucl. Acids Res. 38(19):e181.
[6] Lafaye et al. (2014) DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation. Biochem. Biophys. Res. Commun. 446:341.