We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations.