An Exo-gap method for producing a nested set of unidirectional deletions in a piece of cloned DNA is described. The protein (pII) encoded by gene II of phage f1 makes a single-stranded (ss) nick at the f1 origin of replication (ori) in supercoiled DNA. Many phagemids, such as pBluescriptSK+ contain this ori on one side of the multiple cloning site, thereby permitting purified pII endonuclease to create a nick at one end of a cloned DNA insert. The nick may be expanded into gaps of increasing size by the timed 3' to 5' exonuclease (Exo) activity of the Vent DNA polymerase. Double-stranded deletions are produced by subsequent treatment with ss-specific mung bean nuclease. After size fractionation by agarose-gel electrophoresis, the DNA from the melted gel slices is ligated and transfected into host cells to produce a set of plasmids that contain a unidirectional nested set of deletions. This deletion method is independent of restriction sites, requires only one universal DNA primer to sequence a cloned insert, and may be applied to virtually any cloned segment given the unique nature of the 46-bp recognition site for pII endonuclease.