Isothermal Low Temperature Range

DNA polymerases for point-of-care diagnostic assays

Isothermal Low Temperature Range

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To accommodate the need for high quality enzymes for isothermal amplification ArcticZymes developed the IsoPol® series of polymerases. For isothermal amplification at lower temperatures (37°C and below) we offer two enzymes, IsoPol® DNA Polymerase and IsoPol® SD⁺. Both enzymes exhibit excellent processivity and high strand displacement activity, having 5’-3’ polymerase activity while lacking both 3’-5’and 5’-3’ exonuclease activity.

IsoPol® SD⁺ is an engineered version of IsoPol® DNA Polymerase with even stronger strand displacement and higher salt tolerance.

Temperature
Range

Optimal
Temperature

Strand
displacement
with salt

Salt tolerance
(NaCI/KCL)
(mM)

Suggested applications

IsoPol® SD⁺

20 - 42°C

37°C

++++

100 - 350

MDA/SDA, RPA, HAD

IsoPol® DNA Polymerase

20 - 42°C

37°C

25 - 110

MDA/SDA, RPA, HAD

Figures

Figure 1. Temperature optimum of IsoPol® SD+

Figure 1. Temperature optimum of IsoPol® SD⁺

The enzyme was assayed at the indicated temperatures 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. Samples were incubated for 1 and 5 minutes. Activity was determined measuring radioactive decay [CPM] of incorporated [³H]-dTTPs in acid insoluble material. ΔCPM/min between time point one and two were used as measure of activity. Results are depicted as relative to maximum. The dashed line represents a stipulation of curve progression based on IsoPol^® DNA Polymerase

Figure 2. Polymerase activity of IsoPol® DNA Polymerase and IsoPol® SD+

Figure 2. Polymerase activity of IsoPol® DNA Polymerase and IsoPol® SD⁺

as function of NaCl concentrations in 10 mM Tris-HCl pH 8.5 (@ 25°C), 5 mM MgCl₂, 0.72 µg/µl denatured herring-sperm DNA, 33 µM dNTPs (incl. [³H]-dTTPs), 0.4 µg/ml IsoPol or IsoPol II (6.5 nM). Incubation temperature/time: 37°C/10 min. Activity determination by counting decay events of incorporated [³H]-dTTPs in acid insoluble DNA product in counts per minute [CPM]. Data represented relative to highest activity of each polymerase. Error bars represent 1x standard deviation of biological replicates.

Figure 3. 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.

Properties

Isopol®

IsoPol® SD⁺

Strand Displacement Activity

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

IsoPol® SD⁺ exhibits stronger strand-displacement activity than other polymerases at 25°C and 37°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.

Optimal performance is achieved in a pH 8.5 buffer supplemented with 4-6 mM MgCl₂ and 100-350 mM salt (NaCl or KCl), at 37°C.

Inactivation

Temperature > 50°C

Isopol®

IsoPol® SD⁺

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.

Specific Activity

Min. 15 000 Units/mg

Min. 7 500 Units/mg

Storage & Stability

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

The enzyme is stable at -20°C for up to 3 years 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. IsoPol® polymerases are is manufactured at our ISO 13485 certified facility in Norway.

Isopol®

IsoPol® SD⁺

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.

25 U IsoPol® SD⁺ 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.

25 U IsoPol® SD⁺ 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.

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Recommended Protocols

Ordering Info

Quantity

Product Name

SKU

Pack Size

Concentration

Price

T7 RNA Polymerase

74100-201

10 000 U

50 U/µl

$ 145.00 USD

Isothermal Low Temperature Range