HighFidelity DNA Polymerase: One-fusion DNA Polymerase Datasheet
- Superior fidelity – about 50x improvement compared to Taq Polymerase
- Excellent performance across a wide range of “difficult” templates
- Long range amplification of complex targets - > 10 kb from genomic DNA
- High speed PCR - reduce reaction times
- dUTP poisoning resistance
- Resistance to blood containing DNA samples (up to 20 % of blood)
Description: One-Fusion DNA Polymerase is a unique artificial enzyme created on the basis of intellectual protein design planning by genetic engineering technique. The enzyme possess high fidelity feature. The processivity of the enzyme is very high, so the combination of processivity with fidelity results in dramatically increased yield of PCR products, very high sensitivity of PCR tests, ability to amplify “difficult” templates.
This dramatic increase in processivity results not only in shorter extension times, but also in more robust amplification and the ability to amplify long templates: really fast.
One-Fusion DNA Polymerase possesses the 5'->3' DNA polymerase activity, 3'->5' exonuclease activity and temperature-depended strand-displacement activity and generates blunt ends in the amplification products.
Unit definition: One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP's into acid-insoluble form in 30 minutes at 75°C under assay conditions: 25 mM TAPS-HCl, pH 9.0 (at 25°C), 100 mM KCl, 1.5 mM MgCl2 , 1 mM Beta-mercaptoethanol, 200 μM each dNTP, and 10 μg activated calf thymus DNA in 50 μl.
Associated Activities: Endonuclease and exonuclease activities were not detectible after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoR I digested lambda DNA, respectively, at 72°C in the presence of 15-20 units of One-Fusion DNA polymerase.
Storage buffer: 20mM Tris-HCl, pH 8.0, 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% Glycerol,0.5% Tween 20, stabilizers
Reaction buffers provided:
Storage: store @ - 20°C
Transport: "blue ice" shipment
Note! It is critical that the One-Fusion DNA polymerase is the last component added to the PCR mixture, since the enzyme exhibits 3'->5' exonuclease activity that can degrade primers in the absence of dNTPs.
* results in 1.5mM Mg2+, as the final concentration.
1. one-fusion Polymerase
5. PCR Additives
* optimal: Tm for the primer pair recommended as Tm of the lower primer, for the standard-oligos <20nt.
Further explanation to the protocol:
NOTE: Do not use lower T denaturation, then 98oC; it can cause problems in PCR (nonspecific amplification, poor yield of PCR product, etc.)
For one-fusion DNA polymerase “Annealing” and “Extension” steps should be combined if:
If primers Tm is about 60-61oC for both primers ones can apply simple formula to determine starting Ta/e point - (Tm of the lower primer +72oC)/2. For most applications, it works fine.
To determine a better Ta/e run gradient amplification.
To avoid nonspecific band formation/smearing during amplification not exceed extension time of 30 seconds and use the highest ramp rate of amplificator ( the ramp rate >4-5oC preferable)
3. Post-PCR amplicon detection:
- If you are using whole blood as a template, after amplification spine-down blood cells debris to avoid its application to the gel for DNA detection.
Note: Do not use excess of the blood (>10%), because the post-PCR debris volume of blood cells is very high, and does not allow to run more than 1-2 gels.
- For One-Fusion polymerase Tm of the primers should be corrected, as +3-5oC, comparing with Taq-based PCR conditions.
- For complex DNA templates (human DNA) strongly recommended to apply extension time as 30 sec/Kb