Affinity capillary electrophoresis of DNA for detection of single-nucleotide polymorphisms and point mutations: Comprehensive study for optimization of the weak affinity

A single-stranded DNA and its point mutant can be separated with affinity capillary electrophoresis (ACE) in which an appropriate ligand DNA is used as a pseudo-stationary affinity phase. In this paper, we systematically examine the effects of ligand sequence, capillary temperature, and cation concentration on the ACE separation quality, which is quantitatively evaluated in terms of resolution and peak height. For fine tuning of the affinity, control of MgCl2 concentration and insertion of a spacer sequence into the ligand DNA are more effective than control of the capillary temperature. For design of the ligand DNA, a simple strategy is proposed, based on the prediction of melting temperature. This strategy was tested with eleven different sample sequences. All of them were separated under the same conditions (250 μM MgCl2 and 25 °C), and nine cases of them had satisfactory separation qualities.