Digest ssDNA, but not dsDNA, in a 3´ to 5´ direction
- Specific: Processively digest ssDNA in a 3´ to 5´ direction without harming dsDNA - short 3´ overhangs in DNA are not digested
- Flexible: Active under a wide variety of buffer conditions and can be added directly into most reaction mixes
- Heat Inactivated: Destroy the enzyme activity by incubating at 80°C for 15 minutes
Exonuclease I digests ssDNA in a 3´ to 5´ direction,1-3 but does not digest dsDNA. Although it requires the presence of magnesium and a free 3´-hydroxyl terminus for activity, it is active under a wide variety of buffer conditions and can be added directly into most reaction mixes. Exonuclease I can be heat-inactivated by incubation at 80°C for 15 minutes.
- Removal of residual ssDNA, including oligos, from reaction mixes.
- Removal of ssDNA from nucleic acid mixtures.
Unit Definition: One unit of Exonuclease I catalyzes the release of 10 nmol of acid-soluble nucleotides from heat-denatured calf thymus DNA in 30 minutes at 37°C under standard assay conditions.
Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.
Quality Control: Exonuclease I is tested in degradation of ssDNA and is free of detectable RNase, endonuclease, and double-stranded exonuclease activities.
- Lehman, I.R. and Nussbaum, A.L. (1964) J. Biol. Chem. 239, 2628.
- Kushner, S.R. et al. (1971) Proc. Natl. Acad. Sci. USA 68, 824.
- Kushner, S.R. et al. (1972) Proc. Natl. Acad. Sci. USA 69, 1366.
Figure 1. Specificity of Exonuclease I for ssDNA. 200 ng of EcoR I-linearized pUC19 dsDNA and 1 µg of a 100-mer ssDNA oligo were mixed in 1X TA Buffer and incubated at 37°C for 20 min in the absence or presence of 10 units of Exonuclease I. As seen on this 1% agarose gel, Exonuclease I completely digested the linear ssDNA oligo, but left the linearized plasmid dsDNA intact. Lane 1, size markers; Lane 2, minus Exo I; Lane 3, plus Exo I.