Exonuclease I, 20,000 U

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.

Applications

• 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.

References

1. Lehman, I.R. and Nussbaum, A.L. (1964) J. Biol. Chem. 239, 2628.
2. Kushner, S.R. et al. (1971) Proc. Natl. Acad. Sci. USA 68, 824.
3. 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.