An important manipulation in molecular genetics is to make ordered deletions into a cloned piece of DNA. The most widely used application of this method is in DNA sequencing. Ordered deletions can also be used in delineating sequences that are important for the function of a gene, such as those required for transcription. The principle behind using deletions for sequencing is that consecutive parts of a fragment cloned into a plasmid vector are brought adjacent to a sequencing primer site in the vector. Deletions are generated by digesting DNA unidirectionally with Escherichia coli exonuclease III (ExoIII) (1 ). ExoIII digests one strand of double-stranded DNA by removing nucleotides from 3′ ends if the end is blunt or has a 5′ protrusion. A 3′ protrusion of 4 bases or more is resistant to ExoIII digestion.