FAQ: What is the activity of T7 Endonuclease I on various DNA substrates?

Heteroduplex DNA requires 10-30 units/μg for 20-30 minutes. Double stranded DNA is nicked or cut 1 or 2 bases 5' of mismatches, leaving a 5' PO4 and a 3' OH. Double stranded appears to be cut 50%-70% with the rest being nicked. Linear single stranded DNA is cleaved quickly if it folds back on itself. Small loops (4-15 bases) are cleaved especially if the buffer is optimized using Mn instead of Mg. It may cut misaligned primers. Supercoiled plasmids are more resistant to nicking. Nicking can be seen with 20 units/μg incubated for 4 hours or longer.