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Updated: May 1, 2026
An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Published on: November 29, 2016
Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5'-terminal Cy5 dyes
Tom Killelea1, Christine Saint-Pierre, Céline Ralec
1IFREMER, 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.
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.
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