Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type.