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.

Recognition sequences:

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

Reaction conditions:

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

References

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