A novel system for the rapid generation of precise DNA deletions

To generate DNA deletions, a tandem array of class IIS restriction enzyme recognition sites was cloned into a plasmid. The recognition sites were arranged so that each enzyme cleaves at a different site within an adjacent target sequence. Digestion with both enzymes followed by end repair and ligation resulted in the deletion of DNA between the two sites of cleavage. Because both recognition sites are preserved following deletion, it was found that sequential deletions could be generated using cycles of restriction enzyme digestion, end repair and ligation. Therefore, this system represents a valuable tool in the definition of functional DNA sequences.