AKA: relaxed specificity, altered specificity
England Biolabs Technical Literature
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:
Apo I (2), Ase I (2), BamH I (3), BssH II (2),
EcoR I (4), EcoR V (5), Hind III (1), Hinf I (6,7),
Pst I (8), Pvu II (9), Sal I (8), Sca
I (2), Taq I (10), Xmn I (2).
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
(10). Early studies with EcoR I by Polisky et
al. (4) demonstrated that under conditions of
elevated pH and low ionic strength, EcoR I cleaves
the sequence N/AATTN, while more recent studies
by Gardner et al. (11) showed that EcoR I* (EcoR
I 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.
SgrA I, 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, SgrA I is capable of cleaving non-cognate
sites CRCCGGYN and CRCCGGGG (referred to as secondary
sites). Studies performed with SgrA I 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
Star activity is completely controllable in
the vast majority of cases and is generally not
a concern when performing restriction endonuclease
digests. New England Biolabs enzymes will
not exhibit star activity when used under recommended
conditions in their supplied NEBuffers. Listed
below are reaction conditions known to induce
or inhibit star activity.
Conditions that Contribute
to Star Activity
- High glycerol concentration [>5% v/v]
- High units to µg of DNA ratio [Varies with
each enzyme, usually >100 units/µg]
- Low ionic strength [<25 mM]
- High pH [>pH 8.0]
- Presence of organic solvents [DMSO, ethanol
(9), ethylene glycol, dimethylacetamide, dimethylformamide,
- 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, EcoR I is much
more sensitive to elevated glycerol concentrations
than is Pst I, which is more sensitive to elevated
Inhibiting Star Activity
Recently, 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.
- Use as few units as possible to get a complete
digestion. This avoids overdigestion and reduces
the final glycerol concentration in the reaction.
- Make sure the reaction is free of any organic
solvents such as alcohols which might be present
in the DNA preparation.
- Raise the ionic strength of the reaction
buffer to 100-150 mM (provided the enzyme is
not inhibited by high salt).
- Lower the pH of the reaction buffer to pH
- Use Mg++ as the divalent cation.
- Nasri, M. and Thomas, D. (1986) Nucleic
Acids Res. 14, 811.
- New England Biolabs (unpublished observations)
- George, J., Blakesley, R. W. and Chirikjian,
J. G. (1980) J. Biol. Chem. 255, 6521.
- Polisky, B. et al. (1975) Proc. Natl. Acad.
Sci USA 72, 3310.
- Kuzmin, N. P. et al. (1984) Mol.
Biol (Moscow) 18, 197.
- Petronzio, T. and Schildkraut, I. (1990) Nucl.
Acids Res. 18, 3666.
- Kriss, J. et al. (1990) Nucleic Acids Res.
- Malyguine, E., Vannier, P., Yot. (1980) Gene
- Nasri, M. and Thomas, D. (1987) Nucleic
Acids Res. 15, 7677.
- Barany, F. (1988) Gene 65, 149.
- Gardner, R. C., Howarth, A. J., Messing, J.
and Shepherd, R. J. (1982) DNA 1, 109.
- Tikchinenko, T. I. et al. (1978) Nucleic
Acids Res. 4, 195.
- Bitinaite, J. and Schildkraut, I. (2002) Proc.
Natl. Acad. Sci USA 99, 1164-1169.