A discontinuous buffer system increasing resolution and reproducibility in DNA sequencing on high voltage horizontal ultrathin-layer electrophoresis.

Abstract

A novel discontinuous buffer system for DNA sequencing based on horizontal ultrathin-layer gel electrophoresis is described. The optimized system, named unbuffered stacking gel/discontinuous borate EDTA-buffer system, is composed of a 0.5 mm thick stacking gel, where standard sequencing reactions (1 microL volume) are easily loaded, and a 50 microns ultrathin running gel, where DNA fragments are separated. The novel discontinuous buffer system allows for sample concentration and efficient injection from the stacking gel into the capillary slab gel. Increased resolution, assessed by autoradiography, can be achieved within 25 min running time already over a 10.1 cm distance from the gel slot compared to the conventional gel system. An advantage of the new system is the capacity to resolve compressions in GC-rich regions, usually causing migrating artifacts in standard gels. The described system affords a major improvement in speed, resolution and reproducibility in DNA sequencing.