Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine

Lirui Guan, Godfried W. van der Heijden, Alex Bortvin, Marc M. Greenberg

Research output: Contribution to journalArticlepeer-review


5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo - and DNA polymerase β. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.

Original languageEnglish (US)
Pages (from-to)2184-2190
Number of pages7
Issue number14
StatePublished - Sep 19 2011


  • DNA demethylation
  • DNA methylation
  • DNA replication
  • Modified nucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry


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