Bleomycin DNA damage: Anomalous mobility of 3'-phosphoglycolate termini in an automated capillary DNA sequencer.

ABSTRACT

An automated capillary DNA sequencer with laser-induced fluorescence detection can be utilised for DNA fragment analysis. The precise mobilities of DNA fragments with different chemical termini are especially important in the determination of the sequence specificity of DNA damaging agents. The aim of this study was to examine the electrophoretic mobility profile of DNA fragments with different 3'-termini. The nature of the 3'-teminal residue was found to have a major effect on the electrophoretic mobility of the DNA fragment, especially for 3'-phosphoglycolate termini that migrated anomalously by 3-6 nucleotides. Using the automated capillary sequencer, the electrophoretic mobilities of DNA fragments with different 3'-termini including 3'-hydrogen, 3'-hydroxyl, 3'-phosphate, and 3'-phosphoglycolate were extensively quantified and compared relative to each other. The 3'-hydrogen termini were generated by dideoxy sequencing; 3'-hydroxyl ends by minus sequencing; 3'-phosphate by Maxam-Gilbert chemical sequencing; and 3'-phosphoglycolate by bleomycin cleavage. The mobilities of these DNA fragments with different 3'-termini were found to be (slowest) 3'-hydroxyl<3'-hydrogen<3'-phosphate<3'-phosphoglycolate (fastest); with average relative mobilities of 0.00<0.12<0.63<4.42 nucleotides, respectively. The possible causes of the unusual electrophoretic mobility of the 3'-phosphoglycolate termini were discussed.