Phusion™ High-Fidelity DNA Polymerase

Description

The unique Phusion™ DNA Polymerase offers performance that no other enzyme can match. The Phusion DNA Polymerase generates templates with an accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates. The unique structure and characteristics of this polymerase make Phusion DNA Polymerase a superior choice for cloning, and sets a new standard for PCR performance: its error rate is 50-fold lower than that of Thermus aquaticus, and 6-fold lower than that of Pyrococcus furiosus - thus being the most accurate of available thermostable polymerases. The Phusion DNA Polymerase possesses processivity 10-fold greater than Pyrococcus furiosus and twice that of Thermus aquaticus.

The Phusion™ Technology

Incorporating an exciting new fusion protein technology, Phusion 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 schematic structure of Phusion™ High-Fidelity DNA Polymerase. 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.

Advantages

Extreme fidelity - A New Standard
Using a lacI-based method modified from previous studies, the error rate of Phusion DNA Polymerase is determined to be 4.4 x 10^-7 in Phusion™ HF Buffer, which is approximately 50-fold lower than that of Thermus aquaticus, and 6-fold lower than Pyrococcus furiosus. Phusion DNA Polymerase exhibits the lowest error rate thus making it the new standard for high-fidelity PCR.

Figure 2. Error rates of different DNA polymerases. Fidelity assays were done using lacI-based method modified from Frey & Suppmann, 1995.

Extreme processivity
The Phusion DNA Polymerase has the highest processivity of all thermostable DNA polymerases tested.

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 speed and yield
The fusion of a double-strand DNA-binding domain to Phusion DNA Polymerase multiplies its 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. A 3.8 kb human genomic DNA fragment was amplified with various polymerases using extension times ranging from 1 min to 7 min 40 sec (see figure 3 below). Phusion DNA Polymerase gave strong specific bands even with the shortest extension time, completing the 3.8 kb fragment with only a one minute combined annealing and extension step. Enzyme amounts also indicate that significantly less of the highly processive Phusion DNA Polymerase is required to complete the task.

Figure 3. A 3.8 kb human beta globin gene was amplified according to supplier’s recommendations using varying extension times.

Extreme robustness - minimize reaction failures
The random amplification of complex genomic Thermus species library illustrates the robust performance of Phusion DNA Polymerase. Phusion DNA Polymerase amplified 15 of the 16 randomly selected amplicons (94 %) with high yields. The success rate of Pyrococcus furiosus was 56 % and Thermus aquaticus 62 % with noticeably lower yields. Because of its robust performance, Phusion 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.

1. Phusion DNA Polymerases generate blunt end PCR products.
   Click here for cloning instructions for Phusion DNA Polymerases
2. Detergent-free buffers are available separately for microarray and DHPLC applications, where PCR products are recommended to be free of detergents.
   Click here for more information on Polymerase Buffers
3. For site directed mutagenesis, Finnzymes offers a complete kit:
   Phusion™ Site-Directed Mutagenesis Kit

Applications

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

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

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

Amplification of whole human mitochondrial DNA
Read more

DHPLC and Microarray applications
Detergent-free reaction buffers are available for Phusion DNA Polymerase. PCR products used for DHPLC analysis are recommended to be free of detergents. Detergents may also cause foaming when PCR products are spotted on microarray slides.

Contents

Phusion 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, stabilizers, 200 µg/ml BSA and 50 % glycerol.

Storage stability
Stable for 1 year from the assay date. Recommended storage temperature -20 °C.

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