Exonuclease I
Exonuclease I
E. coli Exonuclease I is a 3' to 5' exonuclease for single-stranded DNA degradation, resulting in the production of 5'-phosphate mononucleotides from the 3'-hydroxyl termini of single-stranded DNA. This 3' to 5' exonuclease is highly specific for single-stranded DNA and does not react with double-stranded DNA or RNA. Exonuclease I is inactivated by heat treatment at 80°C for 15 minutes.
Applications
- Single-stranded DNA degradation
- Removal of ssDNA fragments in a reaction mixture
- PCR primer digestion post-reaction
Notes
Exonuclease I should not be used for DNA end blunting; short DNA ends are not an appropriate substrate for Exonuclease I. For DNA end blunting, use DNA Polymerase I, Klenow Fragment (2140A, 2140AK, 2140B, 2140BK) or Mung Bean Nuclease (2420A, 2420B) instead.
Buffer
Supplied with 1 ml 10X Exonuclease I Buffer [670 mM Glycine-KOH, pH 9.5, 10 mM DTT, 67 mM MgCl2].
Application: Degradation of ssDNA (100-mer) using Exonuclease I
E. coli Exonuclease I is a 3'→5' exonuclease specific for single-stranded DNA. This enzyme does not digest double-stranded DNA or RNA. To demonstrate this specificity, single-stranded DNA (ssDNA, 100-mer) and a double-stranded DNA fragment from pBR322 were incubated with different amounts of Exonuclease I (10–50 units). The digestion products were then analyzed on an agarose gel.
| Reaction conditions: | |
| ssDNA (100-mer) | 2.5 µg |
| pBR322-EcoR I fragment | 0.5 µg |
| 10X Exonuclease I Buffer | 2 µl |
| Exonuclease I | 10-50 units |
| Sterile distilled water | to 20 µl |
| Total | 20 µl |
Reactions were incubated at 37°C for 30 minutes.
Results: The ssDNA was digested by Exonuclease I, but the pBR322 fragment remained intact.
Conclusion: Exonuclease I digests only ssDNA.
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Notes
Exonuclease I should not be used for DNA end blunting; short DNA ends are not an appropriate substrate for Exonuclease I. For DNA end blunting, use DNA Polymerase I, Klenow Fragment (2140A, 2140AK, 2140B, 2140BK) or Mung Bean Nuclease (2420A, 2420B) instead.
Buffer
Supplied with 1 ml 10X Exonuclease I Buffer [670 mM Glycine-KOH, pH 9.5, 10 mM DTT, 67 mM MgCl2].
Application: Degradation of ssDNA (100-mer) using Exonuclease I
E. coli Exonuclease I is a 3'→5' exonuclease specific for single-stranded DNA. This enzyme does not digest double-stranded DNA or RNA. To demonstrate this specificity, single-stranded DNA (ssDNA, 100-mer) and a double-stranded DNA fragment from pBR322 were incubated with different amounts of Exonuclease I (10–50 units). The digestion products were then analyzed on an agarose gel.
| Reaction conditions: | |
| ssDNA (100-mer) | 2.5 µg |
| pBR322-EcoR I fragment | 0.5 µg |
| 10X Exonuclease I Buffer | 2 µl |
| Exonuclease I | 10-50 units |
| Sterile distilled water | to 20 µl |
| Total | 20 µl |
Reactions were incubated at 37°C for 30 minutes.
Results: The ssDNA was digested by Exonuclease I, but the pBR322 fragment remained intact.
Conclusion: Exonuclease I digests only ssDNA.
