Exonuclease I (Exo I)

- Thermal inactivation at 80°C in 15 min
- Recombinant enzyme

Catalog#	Size, concentration	Supplied with:	Certificate of Analysis	MSDS
Catalog#	Size, concentration	10X Reaction Buffer	Certificate of Analysis	MSDS
EN0581	4000 u (20 u/µl)	1.00 ml	EN0581
EN0582	20,000 u (20 u/µl)	5 x 1.00 ml	EN0582

Product information

Features

Active in PCR buffers.

Applications

Primer removal from PCR mixtures:
– prior to PCR product sequencing (2),
– for one-tube "megaprimer" PCR mutagenesis (3).
Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures.
Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminus (4).

Description

Exonuclease I (ExoI) degrades single-stranded DNA in a 3'=>5' direction, releasing deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaving 5'-terminal dinucleotides intact, see Fig.1.

It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups (1).

Source

E.coli cells with a cloned E.coli sbcB gene.

Molecular Weight

54.5 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the release of 10 nmol of acid soluble nucleotides in 30 min at 37°C.

Enzyme activity is assayed in the following mixture: 67 mM glycine-KOH (pH 9.5), 6.7 mM MgCl₂, 1 mM DTT and 0.17 mg/ml single-stranded [³H]-DNA.

Storage Buffer

The enzyme is supplied in:
20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer

670 mM glycine-KOH (pH 9.5 at 25°C), 67 mM MgCl₂, 10 mM DTT.

Quality Control

The absence of endo-, double-stranded exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Inhibition and Inactivation

Inhibitors: 20% (w/v) PEG 8000 (5).
Inactivated by heating at 80°C for 15 min.

Note

The enzyme is not suitable for removing 3'-overhangs of dsDNA.

Figure 1. Exonuclease I activity.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

PCR Product Clean-Up Prior to Sequencing

The clean-up reaction removes unincorporated primers and degrades unincorporated nucleotides. The resulting PCR product is ready to use for sequencing without additional purification, e.g. using column purification kits.

Prepare the following reaction mixture:

PCR mixture (directly after completion of PCR)	5 µl
Exonuclease I (Exo I)	0.5 µl (10 u)
FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase (SAP)	1 µl (1 u)

Mix well and incubate at 37°C for 15 min.
Stop the reaction by heating the mixture at 85°C for 15 min.

Note

Up to 5 µl of purified PCR products can be used directly for DNA sequencing without further purification.
For reliable sequencing results there should not be non-specific PCR products.
The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA polymerase or polymerase mix.
The procedure is not recommended for downstream cloning applications.

Reference

Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.

References

Lehman, I.R., Nussbaum, A.L., The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase), J. Biol. Chem., 239, 2628-2636, 1964.
Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
Nabavi, S., Nazar, R.N., Simplified one-tube “megaprimer” polymerase chain reaction mutagenesis, Anal Biochem., 2, 346-348, 2005.
Rosamond, J., et al., Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca²⁺, J. Biol. Chem., 254, 8646-8652, 1979.
Sasaki, Y., Miyoshi, D. and Sugimoto, N., Regulation of DNA nucleases by molecular crowding., Nucleic Acids Res., 35, 4086-4093, 2007.

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