Practice question

Star activity refers to loss of specificity where a restriction enzyme cleaves at secondary sequences resembling its canonical site under non-optimal conditions. Fidelity depends on ionic strength, pH, cofactors and glycerol concentration. Elevated glycerol above 5%, high pH above 8.0, low ionic strength, prolonged incubation and high enzyme to DNA ratio relax sequence discrimination. High glycerol and alkaline pH alter enzyme conformation and water activity in the catalytic pocket, permitting cleavage at N* non-canonical positions. Preventing star activity requires using recommended buffer, limiting enzyme volume and maintaining proper reaction conditions.