LENGTH-INDEPENDENT SEPARATION OF DNA RESTRICTION FRAGMENTS IN 2-DIMENSIONAL GEL-ELECTROPHORESIS

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60.degree. C in a concentration gradient of a urea-formamide mixture, each EcoRI fragment of [phage] .lambda. or Escherichia coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in an order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment was retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a 2-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the EcoRI digest of E. coli DNA. Corresponding fragments from an isogenic .lambda. lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.