HighEnd and exclusive Polymerase: faster speed, more accuracy, better results
Features:
– 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)
Applications:
– High Fidelity (Hi-Fi) PCR
– Cloning
– “Hi–Fi” – LD PCR
– “anticontaminated” PCR
– “direct blood” PCR
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 mMBeta-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: The product is supplied with 2,5x Reactionbuffer containing 3,75 mM MgCl2
Storage: store @ – 20°C
Transport: “blue ice” shipment
| Product | Description |
| S9450 | one-fusion high-speed-fidelity Polymerase 200 units |
| S9455 | one-fusion high-speed-fidelity Polymerase 1000 units |
General protocol:
The optimal reaction conditions for One–Fusion DNA Polymerase may differ from PCR protocols for standard (Taq-like) DNA polymerases.
PCR conditions for one–Fusion DNA Polymerase is more similar in PCR conditions to “Phusion-like” DNA polymerases, e.g. the enzyme works better at elevated denaturation and annealing temperatures.
PCR reactions should be set up on ice. Prepare a master mix for the appropriate number of samples to be amplified.
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.
| Component | 50µl reactions | 25µl reactions | Final concentration |
| PCR grade Water | Up to 50 µl | Up to 25 µl | |
| 2.5x One–Fusion Buffer | 20 µl | 10 µl | 1X |
| 10 mM MIX dNTPs | 1 µl | 0.5 µl | 0.2 mM each |
| Primers | 0.3-0.5 mM each | ||
| Template DNA | optionally | optionally | |
| One–Fusion polymerase (2 U/µl)* | 1 µl | 0.5 µl | 0.02 U/µL |
* results in 1.5mM Mg2+, as the final concentration.
In some cases we recommend to optimize Mg concentration in the range 1.5-2.5mM´.
We recommend to use 50µl reaction for the PCR with One–Fusion polymerase.
Datasheet
Stability Test Report
Material Safety Datasheet
Reaction components:
1. one–fusion Polymerase
An optimal amount of enzyme in 50µl reaction is 1U. In some cases it could be reduced up to 0.5U per reaction, depending on the length and complicity of amplified DNA sequence. For non-complex amplicons with the length less than 500bp and GC-content <60% amount of HF-Fuzz could be decreased up to 0.5-1.25U per 50mlreaction.
2. Buffer
2.5X Uni Buffers provides very high reproducibility across the wide range of amplification conditions, including “fast-PCR” (reduced time of PCR reaction). 2,5X Uni Buffer contains 1.5mM Mg2+, as the final concentration. In some cases we recommend to optimize Mg concentration in the range 1.5-4.5mM
3. DNTP’s
For most of applications 200mM of each of dNTP’s as final concentration is an optimal. It’s not necessary to optimize dNTP’s concentration. dUTP or other dUTP derivatives should be used replacing TTP in PCR reaction for “anti-contamination” PCR.
4. Primers
Usually 10-20pmol of each specific primer in reaction is enough to get good PCR result. If you are using 2-step PCR with the whole blood as a template, it’s better to use >= 20pmol of each primer.
5. PCR Additives
One–fusion polymerase is compatible with the most of commonly used PCR additives for enhancing of high GC-content DNA templates (glycerol, betaine, DMSO and other). If one will use any additives; take into account the changes of Tm of primers and DNA to correct annealing temperature.
Cycling:
| Cycle step | 2-step amplification | 3-step amplification | Cycles | ||
| ToC | Time | ToC | Time | ||
| Initial Denaturation | 98oC | 1-5 min | 98oC | 1-5 min | 1 |
| Denaturation Annealing Extension | 98oC-72oC | 2-10 S – 15-30 S/Kb | 98oC 55-72°C * 72oC | 2-10 S 10-30 S 15-30 S/Kb** | 25-35 |
| Final extension | 72oC 4oC | 1-2 min hold | 72oC 4oC | 1-3 min hold | 1 |
* optimal: Tm for the primer pair recommended as Tm of the lower primer, for the standard-oligos <20nt.
** For non-complex DNA templates (plasmid DNA, phage DNA, BAC clone) extension time could be reduced up to 15 sec/Kb
1. Denaturation:
a) Initial denaturation for 5 min at 98oC is necessary only for blood cells lysis;
b) for most applications, including “direct-blood” 5 sec at 98oC is enough for denaturation of the sample in during PCR run
NOTE: Do not use lower T denaturation, then 98oC; it can cause problems in PCR (nonspecific amplification, poor yield of PCR product, etc.)
2. Annealing/Extension:
For one–fusion DNA polymerase “Annealing” and “Extension” steps should be combined if:
-Tm of both primers are not differs dramatically (<3oC);
-Tm of the primers are >65oC (optimal Tm for the primers lays between 65-70oC)
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.
Recommendations:
– For complex DNA templates (human DNA) strongly recommended to apply Extension time as 30 sec/Kb for templates > 1, 5 Kb.
– 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