Separation of random fragments of DNA according to properties of their sequences.

The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.