1. Rapid: the reaction can be completed in 5~15 min.
2. 37 ℃ is the most suitable reaction temperature.
3. Affected by CpG methylation, complete sequence overlap and digestion blocking.
4. Affected by Dcm methylation, complete sequence overlap and digestion blocking.
5. Affected by EcoBl methylation, sequences may overlap and digestion blocking.
6. Enzyme inactivation after 20 minutes of incubation at 80 ℃.
7. 3-hour incubation did not show star activity, and delayed enzyme digestion may show star activity.