T7 Endonuclease I recognizes and cuts mismatched DNA, cruciform DNA, Holliday structures or junctions, and heteroduplex DNA. It can also recognize and cut double-stranded DNA with nicks with a lower speed. The enzyme cuts the first, second or third phosphodiester bond that is 5′ to the mismatch. Recombinant T7 Endonuclease I is purified from E.coli expression system. It has high purity and can be applicable for gene mutation, SNP and TALEN or CRISPR/Cas9-mediated mutation detection. It can recognize DNA mismatches, resolve four-way junction or branched DNA, detect heteroduplex or nicked DNA and randomly cleave linear DNA for shot-gun cloning.