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What causes star activity in restriction enzyme digestions?
Posted July 6, 2023
Bioconjugation Chemical ReagentsClassic DyesDNAEnzymesPhysiological Probes
Answer
Star activity in restriction enzyme digestion refers to the altered digestion specificity of the enzyme, which typically occurs under sub-optimal enzyme conditions. Under non-standard reaction conditions, some restriction enzymes are able to cleave sequences that are similar but not identical to their defined recognition sequence. This results in cleavage of DNA at non-specific sites. 
Some of the sub-optimal conditions that result in star activity restriction enzyme digestion include: 
  • High glycerol concentration of >5%
  • pH higher than 8.0
  • High enzyme concentration of >100 units/mg of DNA
  • Presence of organic solvents such as ethylene glycol, sulphalane, DMSO, dimethylformamide, dimethylacetamide, and others in the reaction mixture 
  • Incorrect buffer or cofactor
  • Prolonged reaction time
Additional resources
Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEMRestriction Enzymes Cut Sites Reference Table Enzymes6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

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