Miniaturized system for rapid field inversion gel electrophoresis of DNA with real-time whole-gel detection.
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abstract
Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (approximately 10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce 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 also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the 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.
