Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′-terminal Cy5 dyes

Tom Killelea 1 Christine Saint-Pierre 2 Céline Ralec 1 Didier Gasparutto 2 Ghislaine Henneke 1, *
* Auteur correspondant
1 LM2E - Laboratoire de microbiologie des environnements extrêmophiles
2 Institut de Nanosciences & de Cryogenie
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.
Keywords : Cyanine 5 dye DNA polymerase assays DNA probes Exonuclease assays Fluorescent-labelled oligodeoxynucleotides Polyacrylamide-urea gel electrophoresis
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Article dans une revue
ELECTROPHORESIS, Wiley-VCH Verlag, 2014, 35 (14), pp.1938-1946. 〈10.1002/elps.201400018〉
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Soumis le : vendredi 30 janvier 2015 - 14:30:52
Dernière modification le : jeudi 11 janvier 2018 - 06:16:39

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Tom Killelea, Christine Saint-Pierre, Céline Ralec, Didier Gasparutto, Ghislaine Henneke. Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′-terminal Cy5 dyes. ELECTROPHORESIS, Wiley-VCH Verlag, 2014, 35 (14), pp.1938-1946. 〈10.1002/elps.201400018〉. 〈hal-01111501〉

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