A method was devised for generating nested deletions in DNA that exploits the difference in frequency of restriction sites recognized by compatible restriction endonucleases. A cloning vector was constructed that contains no common blunt-end or RsaI restriction sites and two 8-bp blunt-end restriction sites flanking a commodious multiple cloning site. DNA fragments are cloned into the multiple cloning site using blue-white selection, and nested deletions are generated by digesting the resulting plasmid with either SwaI or PmeI and partially digesting the insert DNA with RsaI. The DNAs are ligated and transformed, producing afamily of plasmids with different-sized deletions. The DNA sequence of these inserts can be rapidly determined, and the overlapping sequences can be assembled in silico to produce a large DNA contig. Nested deletions generated in this manner can also be used for the structure-function analysis of proteins.