A novel procedure to cleave DNA molecules at any desired base sequence is presented. This procedure is based upon our finding that double-stranded DNA molecules at a site where RecA-mediated triple-stranded DNA structure with a complimentary deoxyoligonucleotide is located can be cleaved by a single-strand specific nuclease, such as nuclease S1 or BAL31, between the first base at the 5' termini of the deoxyoligonucleotides and the nearest base proximal to the 5' termini. Accordingly, the sequence as well as the number of the cleavage sites to be cleaved can be custom designed by selecting deoxyoligonucleotides with specific base sequences for triple-stranded DNA formation. The basic characteristics of the cleavage reaction and typical applications of the procedure are presented with actual results, including those which involve cleavage of complex genomic DNA at the very sites one desires.