Although T7 Endonuclease I has activity on mismatched DNA, it is not an ideal enzyme for cleaving all single base pair mismatches in heteroduplex DNA. Depending on the mismatches in your experiment, you may observe different cleavage efficiencies (Guan, C. et al. (2004) Biochemistry, 43, 4313-4322). T7 Endonuclease I may not be the best choice for detecting mismatches of unknown composition. However, if your mismatch is known to contain C, you can use this enzyme for your assay. We recommend titrating the enzyme to optimize cleavage.