It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed “star” activity. It has been suggested that star activity may be a general property of restriction endonucleases (1) and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions. Testing at New England Biolabs has confirmed reports in the literature that the following restriction endonucleases can be made to exhibit star activity: BamHI (2), BsiWI (3), BsoBI (3), DpnII (3), EcoRI (4), HgaI (3), PflMI (3), PvuII (5), SalI (6), ScaI (3), SspI (3), Tth111I (3).

The manner in which an enzyme's specificity is altered depends on the enzyme and on the conditions employed to induce the star activity. The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking (7). Early studies with EcoRI by Polisky et al. (4) demonstrated that under conditions of elevated pH and low ionic strength, EcoRI cleaves the sequence N/AATTN, while more recent studies by Gardner et al. (8) showed that EcoRI* (EcoRI star activity) cleaves any site which differs from the canonical recognition sequence by a single base substitution, providing the substitution does not result in an (A) to (T) or a (T) to (A) change in the central (AATT) tetranucleotide sequence.

SgrAI, which recognizes and cleaves the sequence CRCCGGYG, displays a new phenomenon of relaxation of sequence specificity. Under standard reaction conditions and in the presence of its cognate site, SgrAI is capable of cleaving non-cognate sites CRCCGGYN and CRCCGGGG (referred to as secondary sites). Studies performed with SgrAI reveal that DNA termini generated by cleaving the cognate site are an essential factor in the cleavage of secondary sites, as the secondary sites are not cleaved on DNA substrates that lack a cognate site (9).

Star activity is completely controllable in the vast majority of cases and is generally not a concern when performing restriction endonuclease digests. The majority of New England Biolabs’ enzymes will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. There are some restriction enzymes that are more prone ot star activity than others. These include ScaI, DpnII, EcoRI and BamHI. Please follow the recommended reaction conditions with these enzymes. Listed below are reaction conditions known to induce or inhibit star activity.

Conditions that Contribute to Star Activity

1. High glycerol concentration [>5% v/v]
2. High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg]
3. Low ionic strength [<25 mM]
4. High pH [>pH 8.0]
5. Presence of organic solvents [DMSO, ethanol (5), ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane (10)]
6. Substitution of Mg⁺⁺ with other divalent cations [Mn⁺⁺, Cu⁺⁺, Co⁺⁺, Zn⁺⁺]

The relative significance of each of these altered conditions is dependent on the enzyme in question. For example, EcoRI is much more sensitive to elevated glycerol concentrations than to elevated pH (2).

Inhibiting Star Activity

There has been much attention given to the fidelity of restriction endonucleases, particularly in forensic applications. If you are concerned about star activity, we recommend the following guidelines.

1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.
2. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation.
3. Using the standard 50 µl reaction volume will prevent evaporation during incubation. Evaporation results in an increased glycerol concentration which contributes to star activity.
4. Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme is not inhibited by high salt).
5. Lower the pH of the reaction buffer to pH 7.0.
6. Use Mg⁺⁺ as the divalent cation.

References:

1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811.
2. George, J., Blakesley, R. W. and Chirikjian, J. G. (1980) J. Biol. Chem. 255, 6521.
3. New England Biolabs (unpublished observations)
4. Polisky, B. et al. (1975) Proc. Natl. Acad. Sci USA 72, 3310.
5. Nasri, M. and Thomas, D. (1987) Nucleic Acids Res. 15, 7677.
6. Malyguine, E., Vannier, P., Yot. (1980) Gene 8, 163.
7. Barany, F. (1988) Gene 65, 149.
8. Gardner, R. C., Howarth, A. J., Messing, J. and Shepherd, R. J. (1982) DNA 1, 109.
9. Bitinaite, J. and Schildkraut, I. (2002) Proc. Natl. Acad. Sci USA 99, 1164-1169.
10. Tikchinenko, T. I. et al. (1978) Nucleic Acids Res. 4, 195.

Please also check these additional information about:
NEBuffer Activity  | High Fidelity (HF) Restriction Enzymes  | Double Digestion | Time-Saver Enzymes