Exonuclease I

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.

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.