pBR 322 has recognition site for more than 30 restriction enzymes and most of them occur once, thus generating a full ­length linear molecule with a cohesive or blunt end. Nine restriction sites are located in the tetracycline gene, whereas ampillicin has sites for only three restriction enzymes.

Thus, cloning in pBR 322 with the aid of anyone of those 12 enzymes will result in insertional inactivation of the ampillicin and tetracycline resistance markers. However, cloning of gene in other sites will not permit the easy selection of recombinants because neither of the antibiotic resistance determinates is inactivated. Normally, when pBR 322 is transfected into E. coli, K12 cells can yield more than IOS transfer or recombinant bacteria.

The transformed bacteria are plated on soluble starch rich medium containing tetracycline. After 24 hours the plates are flooded with indicator solution of iodine and penicillin. The bacteria without insert will produce lactamase (ApT gene) which converts penicillin to penicillonic acid.

Penicillonic acid will then bind to iodine and prevent its action on starch. Then the colony of bacteria without insert will be white whereas the bacteria with insert will be blue in colour as it does not produce lactamase as the application gene is inactivated by insert.

pBR 322 is the most widely used cloning vehicle. In addition, it has been widely used as a model system for study of prokaryotic transcription and translation.