Restriction enzyme overview
- General information about restriction enzymes
- Star activity of restriction enzymes
- Inactivation of restriction enzymes
- Buffer activity with restriction enzymes
- Universal buffers for double digestion with restriction enzymes
- Restriction enzymes affected by methylation
- Methylation-sensitive restriction enzymes
- QC of restriction enzymes
- Ligation cloning overview
- Ligation product guide
Compensating for star activity of restriction enzymes
Under certain reaction conditions, restriction enzymes can lose their specificities to substrate DNA and cleave base sequences that are different from the original recognition sites. This phenomenon is called "star activity", and almost all restriction enzymes can have star activity, depending on enzymes, substrate DNA, and reaction conditions. In addition to relaxation of recognition sites, "nicking activity" (partially cleaved DNA) is also observed.
In order to suppress star activity, we recommend performing reactions at lower glycerol concentrations, neutral pH, and higher salt concentrations. However, these conditions may also create lower reactivity.
The table below lists enzymes with star activity observed under specific experimental conditions. Alternate cleavage sequences are noted where data are available. Keys for the recognition sequence information and reaction conditions are located below the table.
M: A or C
K: G or T
N: A or C or G or T
R: A or G
Y: C or T
W: A or T
S: G or C
A: high levels of glycerol
B: in the presence of Mn2+
C: alkaline pH
D: low pH
E: in the presence of DMSO
F: low ionic strength
G: high ionic strength
H: in the presence of 2-mercaptoethanol
Barany, F. The TaqI 'star' reaction: strand preferences reveal hydrogen-bond donor and acceptor sites in canonical sequence recognition. Gene 65, 149–65 (1988).
Clarke, C.M. and Hartley, B.S. Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus. Biochem. J. 177, 49–62 (1979).
George, J., Blakesley, R. W., Chirikjian, J. G. Sequence-specific endonuclease Bam HI. Effect of hydrophobic reagents on sequence recognition and catalysis. J. Biol. Chem. 255, 6521–4 (1980).
George, J. and Chirikjian, J.G. Sequence-specific endonuclease BamHI: relaxation of sequence recognition. Proc. Natl. Acad. Sci. USA 79, 2432–6 (1982).
Gingeras, T.R. and Brooks, J.E. Cloned restriction/modification system from Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 80, 402–6 (1983).
Grosskopt, R. and Kessler, C., unpublished observations.
Halford, S.E., Lovelady, B.M., Mc Callum, S.A. Relaxed specificity of the EcoRV restriction endonuclease. Gene 41, 173–81 (1986).
Heininger, K., Horz, W., Zachau, H. G., Specificity of cleavage by restriction nuclease from Bacillus subtilis. Gene 1, 291–303 (1977).
Hsu, M. and Berg, P. Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+. Biochemistry 17, 131–8 (1978).
Makula, R.A. and Meagher, R.B. A new restriction endonuclease from the anaerobic bacterium, Desulfovibrio desulfuricans, Norway. Nucl. Acids Res. 8, 3125–31 (1980).
Malyguine, E. Vannier, P., Yot, P. Alteration of the specificity of restriction endonucleases in the presence of organic solvents. Gene 8, 163–77 (1980).
Nasri, M. and Thomas, D. Relaxation of recognition sequence of specific endonuclease HlndIII. Nucl. Acids Res. 14, 811–22 (1986).
Nasri, M., Sayadi, S. Thomas, D. Relaxation of PvuII recognition sequence. FEBS Lett. 185, 101–4 (1985).
Nasri, M. and Thomas, D. Alteration of the specificity of PvuII restriction endonuclease. Nucl. Acids Res. 15, 7677–87 (1987).
Nath, K. and Azzolina, B. A. Cleavage properties of site-specific restriction endonucleases. Gene Amplif. Anal. 1, 113–30 (1981).
Pech, M., Streeck, R.E., Zachau, H.G. Patchwork structure of a bovine satellite DNA. Cell 18, 883–93 (1979).
Polisky, B. et al. Specificity of substrate recognition by the EcoRI restriction endonuclease. Proc. Natl. Acad. Sci. USA 72, 3310–4 (1975).
Shinomiya, T. et al. A new aspect of a restriction endonuclease Tth111 I. It has a degenerated specificity (Tth111 I). J. Biochem. 92, 1823–32 (1982).
Takara Bio Inc., unpublished observations.
Tikchonenko, T.I. et al. EcoRI activity: enzyme modification or activation of accompanying endonuclease? Gene 4, 195–212 (1978).
Woodbury, C.P., Hagenbuchle, O., Von Hippel, P.H. DNA site recognition and reduced specificity of the Eco RI endonuclease. J. Biol. Chem. 255, 11534–46 (1980).
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