Creating Nested DNA Deletions Using Exonuclease III

DNA fragments cloned into plasmids are frequently greater than 500 base pairs in length and thus may be too long to sequence from a single primer-binding site in the vector. An efficient way to sequence such large DNA inserts is to generate a nested set of deletions in the target DNA, effectively moving the priming site closer to the sequence of interest. Similarly, nested deletions can be used to delineate a feature of interest (e.g., a replicon [1 ] or promoter) or to subclone a region of DNA devoid of restriction enzyme sites.