Exonuclease VII is a strict single-strand directed enzyme with 5'→3' and 3'→5' exonuclease activities, making it the only bi-directional exonuclease with single-stranded specificity. Exonuclease VII has no apparent requirement for divalent cations, as it is fully active in the presence of EDTA. Initial reaction products are acid insoluble oligonucleotides which are further hydrolyzed into acid soluble form. The products of limited digestions are small oligomers (dimers to dodecamers).

Properties:
Molecular Weight: xseA subunit = 51.8 kDa; xseB subunit = 8.8 kDa
Optimum Temperature: 37 °C
Optimum pH: 8.0
Inactivation: 95 °C for 10 min.

Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, double-stranded exonucleases, and ribonucleases.

Storage Buffer:
50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.5 mM EDTA, 10 mM DTT, 50% glycerol.

Assay Conditions:
The reaction (50 μL) contains 50 mM Tris-HCl (pH 7.9), 50 mM potassium phosphate (pH 7.6), 8.3 mM EDTA, 10 mM 2-mercaptoethanol, denatured DNA, and enzyme.

Unit Definition:
One unit is the amount of enzyme required to catalyze the conversion of 1 nmol of nucleotide to acid-soluble nucleotide in 30 min at 37 °C under standard assay conditions.

Concentration:
10 units/μL

Source:
E. coli strain containing overproducing clones encoding both the large (xseA) and small (xseB) subunits of Exonuclease VII.

Protocol: Typical Reaction Conditions (50 μL)
70 mM Tris-HCI, pH 8.0
8 mM EDTA
10 mM 2-mercaptoethanol
1 μg DNA
50 μg/mL
0.2 units Exonuclease VII

Incubate at 37 °C for 30 min. Inactivate the enzyme by heating to 95 °C for 10 min. Note that Exonuclease VII is not inhibited by EDTA.

Note: A typical dilution buffer for use with Exonuclease VII is 50 mM Tris, pH 7.9, 1 mM DTT and 0.5 mg/mL acetylated BSA.

Applications:
  1. Mapping positions of introns in genomic DNA.
  2. Removal of vector DNA from inserts with poly (dA-T) tails.
  3. Removal of excess PCR primers.
  4. Removal of single-stranded over-hangs produced by restriction endonucleases.