title: Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5'-terminal Cy5 dyes.
abstract: By using a fluorescent exonuclease assay, we reported unusual electrophoretic mobility of 5'-indocarbo-cyanine 5 (5'-Cy5) labelled DNA fragments in denaturing polyacrylamide gels. Incubation time and enzyme concentration were two parameters involved in the formation of 5'-Cy5-labelled degradation products, while the structure of the substrate was slightly interfering. Replacement of positively charged 5'-Cy5-labelled DNA oligonucleotides (DNA oligos) by electrically neutral 5'-carboxyfluorescein (5'-FAM) labelled DNA oligos abolished the anomalous migration pattern of degradation products. MS analysis demonstrated that anomalously migrating products were in fact 5'-labelled DNA fragments ranging from 1 to 8 nucleotides. Longer 5'-Cy5-labelled DNA fragments migrated at the expected position. Altogether, these data highlighted, for the first time, the influence of the mass/charge ratio of 5'-Cy5-labelled DNA oligos on their electrophoretic mobility. Although obtained by performing 3' to 5' exonuclease assays with the family B DNA polymerase from Pyrococcus abyssi, these observations represent a major concern in DNA technology involving most DNA degrading enzymes.
authors.affiliation: IFREMER, Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, Plouzané, France; Laboratoire de Microbiologie des Environnements Extrêmes, Université de Bretagne Occidentale, UMR 6197, Plouzané, France; CNRS, Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, Plouzané, France.
authors.forename: Tom
authors.initials: T
authors.lastname: Killelea
authors.forename: Christine
authors.initials: C
authors.lastname: Saint-Pierre
authors.forename: Céline
authors.initials: C
authors.lastname: Ralec
authors.forename: Didier
authors.initials: D
authors.lastname: Gasparutto
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Henneke
mesh_terms: D002232:Carbocyanines / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; C085321:cyanine dye 5; D004259:DNA-Directed DNA Polymerase
keywords: Cyanine 5 dye; DNA polymerase assays; DNA probes; Exonuclease assays; Fluorescent-labelled oligodeoxynucleotides; Polyacrylamide-urea gel electrophoresis