Sequence-specific cleavage of DNA by restriction endonucleases has been an indispensable tool in modern molecular biology. However, many potential applications are yet to be realized because of the limited number of naturally available restriction specificities. Efforts to expand this repertoire through protein engineering have met considerable challenges and only brought forth modest success. Taking an alternative approach, we developed a methodology to generate modified DNA susceptible to specific cleavage at selected dinucleotide sequences. This method requires the incorporation of two deoxyribonucleotide analogues by a DNA polymerase: a ribonucleotide and a 5'-amino-2',5'-dideoxyribonucleotide, each of which contains a different base. When linked in a 5' to 3' geometry, the two modified nucleotides act synergistically to promote cleavage at the phosphoramidate linkage, thus providing sequence specificity. Using the transferrin receptor gene as an example, we demonstrate that this dinucleotide cleavage generates discrete DNA fragments that can be either visualized by gel electrophoresis or detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.