The electrophoretic mobility of DNA fragments differing by a single 3'-terminal nucleotide in an automated capillary...

The electrophoretic mobility of DNA fragments that differ by a single 3'-terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5'-fluorescently labelled primer to generate DNA fragments with 3'-hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3'-terminal nucleotide. It was found that the 3'-terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) -C > -A > -T > -G (slower). The C-terminated fragments were the fastest and the G-terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed.