Fractionation of large mammalian DNA restriction fragments using vertical pulsed-field gradient gel electrophoresis

Abstract

A new design for pulsed field gradient (PFG) gel electrophoresis of large (greater than 50 kb) DNA fragments is described. The method eliminates distortion of migration of DNA because of the geometry of the applied electric field, requires a single power supply and a simple switching device, and is extremely simple to use. Parameters investigated include variation in pulse time, conditions of restriction enzyme digestion and DNA concentration, and the use of different restriction endonucleases. The method has been applied to restriction enzyme digested mammalian DNA from Chinese hamster and human/Chinese hamster hybrid sources and has allowed examination of the minimum separation of two DNA markers known to be on the same human chromosome, chromosome 21.