Intracellular Detection of Cytosine Incorporation in Genomic DNA by Using 5‐Ethynyl‐2′‐Deoxycytidine

Abstract 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.