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

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.