T7 Gene 6 Exonuclease

The T7 Gene 6 Exonuclease hydrolyzes duplex DNA non-processively in the 5'→3' direction from both 5'-phosphoryl or 5'-hydroxyl nucleotides by liberating oligonucleotides as well as mononucleotides, until about 50% of the DNA is acid soluble. It also degrades nucleotides at the gaps and nicks of double-stranded DNA from the 5'-termini and RNA from RNA:DNA hybrids in the 5'→3' direction.

The T7 Gene 6 Exonuclease is similar to Lambda Exonuclease in that it catalyzes the stepwise hydrolysis of duplex DNA from the 5' termini liberating 5' mononucleotides. However, unlike Lambda Exonuclease, the enzyme has low processivity and it will remove both 5'-hydroxyl and 5'-phosphoryl termini.

It also degrades RNA and DNA from RNA:DNA hybrids in the 5'→3' direction but is unable to degrade either double-stranded or single-stranded RNA.

This enzyme has been used for generating single-stranded DNA templates for sequencing or SNP analysis.

Properties
Molecular Weight: 32 kDa
Optimum pH: 7.5
Optimum Temperature: 37 °C
Inactivation: 75 °C for 10 min or add 2 μL of 0.5M EDTA for a 50 μL reaction volume.

Inactivation
Heating at 75 °C for 10 min or by adding 2 μL of 0.5M EDTA for a 50 μL reaction volume.

Purity
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases and ribonucleases.

Storage Buffer
50 mM KPO₄ (pH 6.5), 1 mM EDTA, 1 mM DTT, 50% glycerol.

Assay Conditions
The reaction mixture (50 μL) contains 50 mM Tris-HCI (pH 8.1), 5 mM MgCl₂, 20 mM KCI, 5 mM 2-mercaptoethanol, double-stranded DNA, and enzyme. Incubation is at 37°C for 15 min.

Unit Definition
One unit is the amount of enzyme required to release 1 nmol of acid soluble nucleotide in15 min at 37 °C under standard assay conditions.

Concentration
50 units/μL

Source
E. coli strain containing an overproducing clone of the T7 Gene 6 Exonuclease

Functional Test
Conversion of 0.5 pmol of λ DNA to single-stranded half molecules by 75 units of enzyme in 30 min at 37°C. Verification by agarose gel electrophoresis.

Applications:
  1. Controlled stepwise digestion of double-stranded DNA from the 5' termini
  2. Generating ssDNA templates for sequencing or SNP analysis