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Phusion™ High-Fidelity DNA Polymerase and Kits

		Extreme Fidelity and Speed -- Licensed for PCR*

		Advantages
		Extreme Fidelity - Eliminate errors with industry-leading fidelity. High Speed - Reduce extension times dramatically; 5X faster than Pyrococcus furiosus. Robust - Reduce reaction failures and minimize optimization. High Yields - Increase product yields with minimal enzyme amounts.

Ordering Information Click catalog number for specific product information

Phusion™ High-Fidelity DNA Polymerase
F-530S	100 units (2 units/µl)
F-530L	500 units (2 units/µl)
Phusion™ High-Fidelity PCR Master Mix with HF Buffer
F-531S	100 reactions in 50 µl volume
F-531L	500 reactions in 50 µl volume
Phusion™ High-Fidelity PCR Master Mix with GC Buffer
F-532S	100 reactions in 50 µl volume
F-532L	500 reactions in 50 µl volume
Phusion™ High-Fidelity PCR Kit
F-553S	50 reactions in 50 µl volume
F-553L	200 reactions in 50 µl volume
Phusion™ Hot-Start High-Fidelity DNA Polymerase
F-540S	100 units (2 units/µl)
F-540L	500 units (2 units/µl)
Phusion™ Flash High-Fidelity PCR Master Mix
F-548S	100 reactions in 20 µl volume
F-548L	500 reactions in 20 µl volume

Phusion products are developed and manufactured by Finnzymes Oy. and distributed by New England Biolabs (UK) Ltd.

Extreme Performance

Finnzymes' Phusion™ DNA Polymerase offers performance that no other enzyme can match. Getting the most out of your amplification reactions has always been difficult. Optimizing fidelity, specificity, and yield has been virtually impossible with a single enzyme. No longer is there a need to compromise performance. Phusion DNA Polymerase combines the fidelity you demand with unparalleled speed and robustness.

Incorporating an exciting new fusion protein technology developed by Finnzymes Oy in collaboration with MJ Bioworks, Inc, Phusion DNA Polymerase brings together a novel Pyrococcus-like enzyme with a processivity-enhancing domain. The Phusion DNA Polymerase generates PCR products with accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates. Additionally, Phusion DNA Polymerase is capable of amplifying long templates. In the quality control tests of Phusion DNA Polymerase, 7.5 kb genomic DNA and 20 kb λ DNA are routinely amplified. The unique structure and characteristics of Phusion DNA Polymerase make it a superior choice for cloning. The Phusion High-Fidelity DNA Polymerase sets a new standard for PCR performance.

The Phusion™ Technology

Most thermostable polymerases are not inherently processive. Generally they extend only few bases before falling off of the primed template. Due to this dissociative nature, even a short fragment requires numerous polymerase binding events to complete elongation. Inefficient or incomplete elongation results in an overall decrease in reaction efficiency and requires longer extension times and increased enzyme concentrations to ensure template completion. Polymerase performance is further challenged by long or difficult templates, resulting in lower yields and a higher rate of reaction failures.

By fusing a particular double-stranded DNA binding domain to the polymerase, its processivity can 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 amplify long templates in a fraction of the time.

Figure 1. Phusion High-Fidelity DNA Polymerase schematic. The doublestranded DNA binding domain (blue) is fused with a novel Pyrococcus-like enzyme (green) forming a unique high performance polymerase.

Extreme Fidelity - A new standard

The fidelity values of Phusion DNA Polymerase and other DNA polymerases are presented in Figure 2. Using a lacIbased method, 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 DNA polymerase, and 6-fold lower than that of Pyrococcus furiosus DNA polymerase. Phusion DNA Polymerase exhibits the lowest error rate thus making it the new standard for high-fidelity PCR and cloning. Phusion DNA Polymerase produces blunt end PCR products.

Figure 2. Error rates of different DNA polymerases. Fidelity assays were done using a lacI-based method modified from Frey & Suppmann, 1995. The error rate of Phusion DNA Polymerase was 4.4 x 10^-7 in Phusion HF Buffer, which makes Phusion DNA Polymerase the most accurate DNA polymerase available.

Extreme Processivity

Phusion DNA Polymerase has the highest processivity of all thermostable DNA polymerases tested. The processivity of Phusion DNA Polymerase is approximately 10-fold greater than that of Pyrococcus furiosus DNA polymerase and twice that of Thermus aquaticus DNA polymerase.

The relative processivity values of Phusion™ DNA Polymerase and other DNA polymerases
Phusion™ DNA Polymerase	10
Thermococcus kodakaraensis DNA polymerase	8
Pyrococcus furiosus DNA polymerase	1
Thermus aquaticus DNA polymerase	6

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-100) and 200 µM of each dNTP. DNA polymerase was added to the primed template at a molar ratio of ~1:4000 to initiate DNA synthesis at 72°C. Samples taken at various times were diluted in gel loading dye, and analyzed on a sequencer. 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 - Reduced extension and overall cycling times

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. Phusion DNA Polymerase gave strong specific bands even with the shortest extension time, completing the 3.8 kb fragment with a combined annealing and extension step of only 1 minute (total cycling time 1h). DNA polymerase from Pyrococcus furiosus did not amplify any product even with the longest extension time (total cycling time more than 5 hours), and modified Pyrococcus furiosus DNA polymerase amplified a weak band with 3 min 50 s extension time (total cycling time 3 hours). It should also be noted that significantly fewer units of the highly processive Phusion DNA Polymerase were required to complete this task.

Figure 3. A 3.8 kb human beta globin gene was amplified according to suppliers' recommendations using varying extension times. Phusion DNA Polymerase was able to amplify the 3.8 kb genomic fragment with a combined annealing and extension step of only 1 minute, thus being significantly faster than the two other polymerases tested. A single unit of Phusion DNA Polymerase produced superior yields than 2.5 or 5 units of the Pyrococcus furiosus DNA polymerases.

Enzyme amounts/Cycling protocols	Pyrococcus furiosus	modified P. furiosus	Phusion™ DNA Polymerase
Enzyme amount	5 units/50 µl	2.5 units/50 µl	1 units/50 µl
Initial denaturation	95°C 45 s	95°C 2 min	98°C 30 s
Denaturation	95°C 45 s	95°C 30 s	98°C 10 s
Annealing	60°C 45 s	60°C 30 s	– **
Extension a,b,c and d*	72°C x min	72°C x min	72°C x min
Number of cycles	30	30	30

* Extension times: a) 1 min, b) 1 min 30 s, c) 3 min 50 s and d) 7 min 40 s.
** Due to the unique characteristics of Phusion DNA Polymerase, annealing and extension steps were combined with Phusion DNA Polymerase (= two-step PCR).

Extreme Robustness - Minimize reaction failures

A set of 16 clones were randomly selected from a Thermus sp. genomic library and amplified with three different thermostable DNA polymerases. The results highlight the robustness and speed of Phusion DNA Polymerase. Phusion DNA Polymerase was able to amplify 15 of the 16 randomly selected amplicons (94%) with high yields. The success rate of Pyrococcus furiosus DNA polymerase was 56% and Thermus aquaticus DNA polymerase 62% with noticeably lower yields. Extension times and enzyme amounts used with Phusion DNA Polymerase (3 min, 0.4 U/20 µl) were significantly lower than those needed with Pyrococcus furiosus DNA polymerase (10 min, 1 U/ 20 µl) and comparable to those of Thermus aquaticus DNA polymerase (3 min, 0.5 U/20 µl), yet the yields were superior. These results further confirm the improved performance benefits of this novel polymerase.

Phusion™ DNA Polymerase
	• 0.4 units/20 µl rxn • 3 min extension time • 15 of 16 clones amplified
Pyrococcus furiosus DNA polymerase
	• 1.0 units/20 µl rxn • 10 min extension time • 9 of 16 clones amplified
Thermus aquaticus DNA polymerase
	• 0.5 units/20 µl rxn • 3 min extension time • 10 of 16 clones amplified
Figure 4. A random set of 16 clones from a Thermus sp. genomic library was amplified from bacterial colonies. The amplicon size varied between 1-10 kb. Amplifications were done according to suppliers' instructions using same reaction conditions for all 16 amplicons.

And Now High Specificity - with Phusion Hot Start High-Fidelity DNA Polymerase

Ordering Information

Two different buffers are provided with Phusion High-Fidelity DNA Polymerase and Phusion High-Fidelity PCR Kit. HF Buffer is the default buffer for most purposes and high-fidelity applications. GC Buffer can improve the performance of Phusion Polymerase on some difficult or long templates.

Phusion™ High-Fidelity DNA Polymerase
F-530S	100 units (2 units/µl)
F-530L	500 units (2 units/µl)
Phusion™ High-Fidelity PCR Master Mix with HF Buffer
F-531S	100 reactions in 50 µl volume
F-531L	500 reactions in 50 µl volume
Phusion™ High-Fidelity PCR Master Mix with GC Buffer
F-532S	100 reactions in 50 µl volume
F-532L	500 reactions in 50 µl volume
Phusion™ High-Fidelity PCR Kit
F-553S	50 reactions in 50 µl volume
F-553L	200 reactions in 50 µl volume
Phusion™ Hot-Start High-Fidelity DNA Polymerase
F-540S	100 units (2 units/µl)
F-540L	500 units (2 units/µl)
Phusion™ Flash High-Fidelity PCR Master Mix
F-548S	100 reactions in 20 µl volume
F-548L	500 reactions in 20 µl volume
Buffers available separately
F-518S/L	Phusion™ HF Buffer Pack
F-519S/L	Phusion™ GC Buffer Pack
F-520S/L	Detergent-Free Phusion™ HF Buffer Pack
F-521S/L	Detergent-Free Phusion™ GC Buffer Pack

Phusion and DyNAzyme are trademarks of Finnzymes Oy.

* PCR license notice: These products are sold under licensing arrangements of Finnzymes Oy with F.Hoffman- La Roche LTD, Roche Molecular Systems,Inc. and The Perkin-Elmer Corporation. The purchase of these products is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front fee, either by payment to Perkin-Elmer or as purchased, i.e. an authorized thermal cycler.

Notice to purchaser: Limited license. The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents owned by BIO-RAD Laboratories, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product.