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Star activity-nonspecific cleavage at variant sites

Star activity-nonspecific cleavage at variant sites

The most prominent feature of restriction endonucleases is specific cleavage at their recognition sequences. Restriction endonucleases can bind DNA in a sequence-independent manner, but they cleave DNA at specific sequences. Cleavage efficiency, under optimal reaction conditions, is greatly reduced even when there is even a single base change in the recognition sequence for a restriction endonuclease. For example, one base change in an EcoR l (Cat.# R002) site (5'-GAATTC-3' into 5'-TAATTC-3') reduces the cleavage efficiency 105 fold. EcoR V (Cat.# R018) shows more dramatic change in cleavage efficiency.

 

One base change in an EcoR V (Cat.# R018) site (5'-GATATC-3' into 5'-GTTATC-3') reduces the cleavage efficiency as much as 106 fold. Under non-optimal reaction conditions, however, differences in cleavage efficiencies between the original recognition sequence and its variant are not as great as the two examples above. Under these conditions, some restriction endonucleases lower their specific cleavage fidelity; they tend to cleave both specific and variat sequences under non-optimal conditions. This nonspecific cleavage ability of restriction endonucleases is referred to as star activity. The star activity may be a general property of a restriction enzyme, although they differ greatly in the extents of star activity. All restriction endonucleases display some star activity, especially under non-optimal reaction conditions. Restriction endonucleases that display marked star activity in non-optimal reaction conditions include BamH l (Cat.# R003), Bcl l (Cat.# R048), EcoR l (Cat.# R002, Hind lll (Cat.# R008), Kpn l (Cat.# R014) Nde l (Cat.# R006), Pst l (Cat.# R019), Sal l (Cat.# R009), Sph l (Cat.# R017), and Tth111 l (Cat.# R038). Fortunately, star activity is completely controllable and is generally not a problem for most restriction endonucleases. Under recommended reaction conditions, commercially available restriction endonucleases rarely exhibit star activity. To avoid or minimize star activity, however, reaction conditions should not be deviated much from the ones recommended, especially when using the several enzymes that tend to have high star activities.

 

Factors that cause star activity

High concentrations of glycerol (>5% v/v)

High ratios of enzyme to DNA (variable, usually >20-100 units/μg)

Low ionic strength (<25 mM)

Elevated pH (>pH 8.0)

Presence of organic solvents (DMSO, ethanol, ethylene glycol, dimethylacetamide,

dimethylformamide, sulphalane, and so on)

Divalent cations (Mn2+, Cu2+, Co2+, Zn2+) other than Mg2+

 

Conditions to suppress star activity

Reduce the final glycerol concentration by not adding excess restriction endonucleases

Avoid organic solvent contamination during DNA purification

Increase ion strength of reaction buffer to 100-150 mM

Reduce the pH of reaction buffer to 7.0

Use only Mg2+ as the divalent metal ion

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