To quantitatively detect single-base mutations in genetic samples, free-solution affinity capillary electrophoresis was developed using a diblock copolymer probe composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG). When the probe is designed to be complementary to a wild-type single-stranded DNA sample (WT), WT migrates more slowly than its single-base mutant (MT). This is because WT forms a reversible complex with the probe via hybridization during the electrophoresis, resulting in a large amount of hydrodynamic friction. In contrast, a negligibly weak interaction takes place between MT and the probe due to a single-base mismatch. Accordingly, the MT peak appears earlier than the WT peak on the electropherogram. The difference of the migration time between WT and MT depends on the thermodynamic stability of the complex and also on the amount of hydrodynamic friction that the complex encounters. Therefore, the peak resolution is rationally controlled by the basenumber of the ODN segment and by the molecular weight of the PEG segment. Importantly, the peak area ratio provides the occurrence ratio of the single-base mutant within the given population. This facile and accurate method to estimate SNP allele frequency should find applications in various research fields.
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