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Intracellular Detection of Cytosine Incorporation in Genomic DNA by Using 5‐Ethynyl‐2′‐Deoxycytidine
Author(s) -
Guan Lirui,
van der Heijden Godfried W.,
Bortvin Alex,
Greenberg Marc M.
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100353
Subject(s) - klenow fragment , dna polymerase , dna polymerase i , dna demethylation , dna ligase , microbiology and biotechnology , dna polymerase ii , chemistry , dna , dna clamp , dna replication , polymerase , biochemistry , biology , dna methylation , polymerase chain reaction , exonuclease , gene , reverse transcriptase , gene expression
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.