A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e., in a gel containing a constant level of positive charges from anode to cathode). In this last case, separation is still obtained due to differential charge modulation of the various DNA fragments. In the 100-1000-bp length, it is shown that separation can be obtained even for fragments differing in length by <0.5%, as shown in the splitting of a 656- and 659-bp doublet, that could not be resolved by conventional polyacrylamide gels. In the 10-100-bp range, it is shown that the present method can resolve single nucleotide polymorphisms, i.e. fragments of identical number of nucleotides but differing by one base substitution. In this last case, separations are obtained only in gradient gels containing a much steeper gradient of charges (0-20 mM Immobiline pK 10.3 and pK 12, as opposed to gradients of only 2-4 mM positive charges for larger size fragments). This novel methodology represents a marked improvement over existing techniques and appears to hold promises for applications in diverse fields, such as molecular biology, forensic medicine, and genetic screening.