We developed a reliable method for detecting single nucleotide mutation by using spontaneous aggregation of a DNA-carrying colloidal nanoparticle, which was composed of a hydrophobic core from poly(N-isopropylacrylamide) (polyNIPAAm) and a hydrophilic shell from probe DNA. The particle with a diameter of 50 nm was formed through self-assembly of polyNIPAAm-DNA copolymers above the phase transition temperature of polyNIPAAm part. Adding the complementary DNA into the colloidal dispersion, the particles were found to aggregate rapidly in buffer (pH 7.4) containing 0.5 M NaCl through a non-crosslinking mechanism. In contrast, they kept dispersing in the presence of the one point-mutated DNA under the identical conditions. When using capillary electrophoresis (CE), sequence-specific DNA separation was attained based on a difference in affinity of sample DNAs with a probe DNA-containing water-soluble nonionic polymer. By the miniaturization using a microchip made of polydimethylsiloxane, we have succeeded in much faster separation than with the conventional CE apparatus.