A simple strategy to generate small deletions using Bal31 nuclease.

The definition of the functional boundaries of regulatory DNA elements often includes sequential deletion of DNA using controlled digestion with BaBl nuclease [for example, (1, 2)]. To obtain small deletions (< 100 bp) we have devised a simple strategy involving cloning of the target DNA into the EcoSl site of the pBR322 plasmid. The hybrid plasmid is linearized at a unique restriction site present at the 3' end of the target DNA and used as substrate for controlled BaBl digestion. BaBl nuclease will generate deletions of both ends at approximately the same rate (3). Deletion of the vector end larger than 30—50 bp (assuming that the length of insert DNA 3' of the site used for linearization is no more than 20 bp) will inactivate the tetracycline resistance gene of pBR322. Thus, selection for tetracycline resistant transformants will yield deletions of the target DNA in the order of 100 bp or less. Using this experimental approach we obtained sequential deletions of the target DNA ranging 20 to 100 bp in length.