A nuclease is any enzyme which cleaves DNA or RNA by breaking phosphodiester bonds, either from within the molecule (endonucleases) or from either (or both) the 3' and 5' ends of the molecule (exonucleases).

Endonucleases degrade nucleic acids internally from a recognition site in the molecule. They are used to generate fragments for cloning, and to map genes or polymorphisms. Examples of endonucleases include:
  • S1 nuclease, which cleaves single-stranded DNA non-specifically. It is used in S1 nuclease protection to map transcription start sites, or to identify the boundaries between introns and exons. It may be used with exo III (see below) to produce deletions.
  • DNase I, which cleaves double-stranded DNA non-specifically. It is used to destroy DNA and generate short random fragments for cloning or directed evolution methods

Exonucleases degrade nucleic acids one-by-one from either the 3' or 5' end of the molecule (sometimes both). They are commonly used to produce deletions or to trim ends. Examples of exonuclease include:
  • Bal31, which degrades from both 3' and 5' ends
  • ExoIII, which degrades only from the 3' end. It proofreads DNA in the absence of dNTPs, and is used in conjunction with S1 endonuclease to produce deletions.