Determination of genomic 5-hydroxymethyl-2'-deoxycytidine in human DNA by capillary electrophoresis with laser-induced fluorescence

Annette M. Krais, Yoon Jung Park, Christoph Plass, Heinz H. Schmeiser

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


Recent studies reported the presence of 5-hydroxymethylcytosine (5-hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine. Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5-hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5-hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5-hmdC of 0.45 amol, which is equivalent to approximately one 5-hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix. By applying the calibrated assay to the analysis of various DNAs we show that 5-hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.

Original languageEnglish
Pages (from-to)560-565
Number of pages6
Issue number5
StatePublished - May 2011


  • 5-hydroxymethyl-2'-deoxycytidine
  • 5-methylcytosine
  • Capillary electrophoresis
  • DNA methylation
  • Epigenetics
  • Human cancer
  • Laser-induced fluorescence


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