title: 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.
authors.affiliation: Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Lirui
authors.initials: L
authors.lastname: Guan
authors.forename: Godfried W
authors.initials: GW
authors.lastname: van der Heijden
authors.forename: Alex
authors.initials: A
authors.lastname: Bortvin
authors.forename: Marc M
authors.initials: MM
authors.lastname: Greenberg
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D003300:Copper / Q000737:chemistry; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003841:Deoxycytidine / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D016678:Genome / Q000235:genetics*; D042541:Intracellular Space / Q000378:metabolism*; D008297:Male; D051379:Mice; D015336:Molecular Probe Techniques; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D015053:Zygote / Q000166:cytology / Q000378:metabolism
chemical_list: C569100:5-ethynyl-2'-deoxycytidine triphosphate; D000480:Alkynes; D001386:Azides; D003845:Deoxycytosine Nucleotides; D005456:Fluorescent Dyes; D003841:Deoxycytidine; D003300:Copper; D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase