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