Separating field strength, temperature, and pulsing effects in pulsed field electrophoresis

The utility of pulsed field electrophoresis for DNA sequencing is investigated. Previous studies have indicated a beneficial retardation of sequencing fragments when pulsed fields are employed. The interpretation of these results is complicated, however, by concomitant variations in electric field strength and/or temperature. Methods are presented here permitting discrimination of such mobility effects due to pulsing, field strength, and temperature. It is demonstrated that under the conditions employed here, observed mobility effects are due to electric field variations rather than pulsing. These conditions thus correspond to the low frequency/small molecule limit. The effect of temperature is estimated from the steady state solution to the heat conduction equation under appropriate boundary conditions. No temperature effect upon mobility is operative in the thin gel system employed, due to the high efficiency of heat transfer. However, it is shown that in conventional gel systems large temperature-related mobility effects can occur. These methods for dissecting and understanding mobility effects in pulsed field electrophoresis are expected to be of general utility.