Phusion™ Hot Start High-Fidelity DNA Polymerase

The Phusion™ Technology

Incorporating an exciting fusion protein technology, Phusion Hot Start DNA Polymerase brings together a novel Pyrococcus-like enzyme with a processivity-enhancing domain. By fusing a double-strand DNA-binding domain to the polymerase, its processivity can easily be increased 10-fold. Phusion DNA Polymerase exploits this dramatic increase in processivity, resulting in shorter extension times, more robust and high yield amplification, and the ability to do long templates in a fraction of time.

Figure 1. The structure of Phusion™ High-Fidelity DNA Polymerases. The double-strand DNA-binding domain (purple) is fused to a novel Pyrococcus-like enzyme (green) forming a unique high-performance polymerase - Phusion DNA Polymerase.

Extreme fidelity - A New Standard

Figure 2. Relative fidelity values of different DNA polymerases. Fidelity = 1 / error rate. Fidelity assays were done using lacI-based method modified from Frey & Suppmann, 1995.

Extreme processivity

Processivity assay. A 5' FAM-labeled primer was annealed to ssM13mp18 DNA. The primed template was pre-formed in the presence of standard PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 2 mM MgCl₂, and 0.1 % Triton® X -100) and 200 µM of each dNTPs. DNA polymerase was added to the primed template at a molar ratio of ~1:4,000 to initiate DNA synthesis at 72 °C. Samples taken at various times were diluted in gel loading dye, and analyzed on a MJ GeneWorks BaseStation ® (MJ Research). The median product length was determined based on the integration of all detectable primer extension products. When the median product length does not change with an increase in reaction time or a decrease in polymerase concentration, it is used as a measure of processivity.

Extreme specificity

The hot start modification of Phusion Hot Start DNA Polymerase increases the specificity of PCR amplification by preventing non-specific extension of DNA substrate at ambient temperatures prior to the first cycle of PCR.

Extreme speed and yield

The fusion of a double-strand DNA-binding domain to Phusion DNA Polymerases multiplies their processivity in respect of other DNA polymerases. This dramatic increase in processivity results in shorter extension times, more robust and high yield amplification, and the ability to do long templates in a fraction of the time.

For more details on speed and yield, see
Phusion High-Fidelity DNA Polymerase

Extreme robustness

The robustness of Phusion Hot Start DNA Polymerase reduces reaction failures and minimizes optimization. Because of its robust performance, Phusion Hot Start DNA Polymerase is capable of processing templates in the presence of additives such as DMSO, or in reactions containing impurities like debris from cell suspensions.

For more details on robustness, see
Phusion High-Fidelity DNA Polymerase.

Applications

High-fidelity PCR
The error rate of Phusion Hot Start DNA Polymerases is 4.4 x 10^-7, which is industry-leading fidelity. Therefore Phusion Hot StartDNA Polymerase is suitable for all PCR applications requiring great accuracy.

Cloning
Phusion Hot Start DNA Polymerase amplifies templates with an accuracy and speed previously unattainable with a single enzyme. This makes it a superior choice for cloning. Phusion Hot Start DNA Polymerase produces blunt ends to the amplified fragment.

Long amplicons
Extension and overall cycling times can be significantly reduced when using Phusion Hot Start DNA Polymerase. Due to the high processivity Phusion Hot Start DNA Polymerase can amplify long templates in a fraction of the time other polymerases need.

Amplification of whole human mitochondrial DNA
Read more

Contents

Phusion Hot Start High-Fidelity DNA Polymerase is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, DMSO and 50 mM MgCl₂ solution. Both Phusion HF Buffer and Phusion GC Buffer contain 7.5 mM MgCl₂ in the provided 5x concentration.

Storage buffer
20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 200 µg/ml BSA and 50% glycerol.

Storage and stability
Recommended storage temperature -20°C. Stable for one year from the assay date.

Ordering information