Mobility and Diffusion Regimes In Field Inversion Gel Electrophoresis of DNA In the Sub-35 Kilobase Size Range

Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications, butut many aspects of DNA migration phenomena under these conditions are not well understood. One of the main reasons for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (~ 10 - 15 hours) and the need to perform gel analysis by post-staining after completion of the run. We have developed an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60 - 90 min. The compact size of the device combined with the use of quartz as the substrate material permit the gel to be continuously illuminated with UV light so that the separation processes can be recorded in real time using a CCD camera.

These capabilities allow us to probe size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings, and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields.