Common Applications for Exonucleases and Endonucleases

Application Recommended Enzyme(s) Notes
Removal of 3′ overhangs
5′ overhang Fill in Treatment
Removal of single-stranded primers for nested PCR reactions
Removal of primers post PCR prior to DNA sequencing or SNP detection
  • Quick Heat inactivation versus Exonuclease I For 3′ chemically modified primers
  • Quick Heat inactivation versus Exonuclease I for 3′chemically modified primers
Mapping positions of introns in genomic DNA
Removal of primers with or without 3′ or 5′ terminal phosphorothioate bonds

Generating ssDNA from linear dsDNA:

  • If 5′ → 3′ polarity required
  • If 3′ → 5′ polarity required


  • Strand targeted for removal requires one 5′ end with phosphate
  • Strand targeted for removal requires a 5′ overhang, a blunt end, or a 3′ overhang (with less than 4 bases)
Preparation of nested deletions in double-stranded DNA
Site-directed mutagenesis
  • Removes nicked-strand DNA from 3′ to 5′
  • Removes nicked-strand DNA from 5′ to 3′
Nick-site extension
Degradation of denatured DNA from alkaline-based plasmid purification methods for improving DNA cloning
Removal of chromosomal/linear DNA in plasmid preparations
  • Degrades linear ss + dsDNA, nicked DNA
  • Degrades linear ss + dsDNA: PREFERRED as Exo V will save nicked plasmids resulting in higher yields especially for low-copy number plasmid prep
Removal of unligated products (linear dsDNA) from ligated circular double-stranded DNA
  • Only the un-nicked form of ligated circular double-stranded remains
  • Both nicked and unnicked-form of ligated circular double-stranded DNA remains
Removal of residual gDNA after purification of low copy plasmid
Removal of contaminated DNA from RNA samples
Removal of template DNA from IVT reactions
Conversion of single-stranded DNA or RNA to 5′-mononucleotides
Analysis of base composition, potential damage and modification of nucleic acids
Preparation of double-stranded DNA fragments with 5′-OH and 3′-phosphate
Degradation of nucleic acids (both DNA and RNA) in crude cell-free extracts
Preparation of rabbit reticulocyte
Chromatin Immunoprecipitation (ChIP) analysis