Applications
- Removal of residual ssDNA, including oligos, from reaction mixes.
- Removal of ssDNA from nucleic acid mixtures.
Exonuclease I digests ssDNA in a 3´→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.
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 doublestranded exonuclease
activities.
References
- 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.
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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. |
