IsoPol™ DNA Polymerase

Overview

IsoPol™ DNA Polymerase is active at ambient temperatures.

It exhibits 5’-3’ polymerase activity and lacks 3’-5’ exonuclease activity. The 5’-3’ exonuclease domain is truncated, thus the enzyme also lacks 5’-3’ exonuclease activity. IsoPol™ DNA Polymerase has excellent processivity and strong stand-displacement activity.

In addition, it can be heat inactivated at temperatures above 50°C.

Figure 1. Activity of IsoPol™ DNA Polymerase incubated for 10 minutes at temperatures from 10 to 50°C. The polymerase was assayed in 10 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM MgCl2 , 700 µg/mL denatured herring sperm DNA, 33 µM dNTPs (each), 0.125 µM [3H]-dTTP, 0.8 mM DTT, 0.02 % Triton X-100.

Properties

Unit Definition: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C. IsoPol™ DNA Polymerase exhibits a specific activity of ~20000 U/mg.

Relative specific activity: The relative specific activity of the IsoPol™ DNA Polymerase was compared to other commercially available polymerases by using assay conditions as mentioned in the unit definition above.

Figure 2: Relative activity of IsoPol™ DNA Polymerase compared to other commercially available polymerases at 37°C. The enzymes were assayed in 10 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM MgCl2 , 700 µg/mL denatured herring sperm DNA, 33 µM dNTPs (each), 0.125 µM [3H]-dTTP, 0.8 mM DTT, 0.02% Triton X-100. Activity of Phi29 polymerase was close to non-detectable in this setup.

Strand-displacement activity: IsoPol™ DNA Polymerase exhibits similar strand-displacement activity as that of Bsu DNA polymerase I at 25°C.

Optimal reaction conditions: Optimal performance is achieved in a pH 8.5 buffer supplemented with 5 mM MgCl2 and 25-100 mM salt (NaCl or KCl), at 37°C.

Storage and Stability

The enzyme is stable at -20°C for > 1 year in the supplied storage buffer. The enzyme tolerates at least five freeze-thaw cycles (-80°C) without loss of activity.

Quality Control

ArcticZymes is dedicated to the quality of our products. We manufacture all our products at our ISO 13485 certified facility in Norway

dsDNA endonuclease activity

25 U IsoPol™ DNA Polymerase was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.

ssDNA endonuclease activity

25 U IsoPol™ DNA Polymerase was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.

Additional Data and Information

We are pleased to provide data and information relating to IsoPol™ DNA Polymerase. The Product is an Early Access Product, and further product information will be generated continuously. Available data includes; buffer and storage conditions, optimal conditions, activity processivity, and strand displacement data.

We appreciate suggestions for product modifications.