AbstractThe transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes fromS. pombeandS. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.