DNase I (RNase-free) is an endonuclease that digests single- or double- stranded DNA, generating dinucleotide, trinucleotide, and oligonucleotide products with 5´-phosphorylated and 3´-hydroxylated ends. The activity of DNase I (RNase-free) dependent on Ca²⁺ and can be activated by Mg²⁺ or Mn²⁺. In the presence of Mg²⁺, DNase I (RNase-free) can randomly cleaves arbitrary sites of double-stranded DNA; in the presence of Mn²⁺, DNase I (RNase-free) can simultaneously recognize both strands of DNA and cleaves at nearly same sites.

DNase I (RNase-free) can act on single stranded DNA, double stranded DNA, chromatin, and RNA: DNA hybrid strands. DNase I (RNase-free) is suitable for RNA extraction, in vitro transcription, and removal of DNA for RT-PCR experiments.