Methods are provided for separating linear nucleic acid fragments based on their conformation. The methods are based on novel two-dimensional gel electrophoresis techniques. In one aspect the method comprises electrophoresing a sample of nucleic acids in a first dimension said sample through a gel matrix under a first set of pre-determined electrophoresis conditions; electrophoresing said gel matrix in a second dimension under a second set of electrophoresis conditions, such that linear nucleic acid fragments of equal length but having different conformation are separated; said first and second electrophoresis conditions are different, such that in one of said dimensions electrophoresis allows separation of linear nucleic acid fragments based on conformation and length, and in the other of said dimensions electrophoresis allows separation of the sample fragments based substantially on length. Said difference is preferably established with a chemical agent capable of reducing conformational differences between linear nucleic acids fragments.