Phusion

Phusion™ High-Fidelity DNA Polymerase

The most accurate of available thermostable polymerases

Description

New unique Phusion DNA polymerase offers performance that no other enzyme can match. The Phusion 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 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 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 developed in collaboration with MJ Bioworks, Inc, 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 structure of Phusion High-Fidelity DNA polymerase. The double-strand DNA binding domain (blue) is fused together with a novel pyrococcus-like enzyme (green) forming a unique high performance polymerase - Phusion DNA polymerase.

Advantages

Extreme fidelity
Extreme processivity
Extreme speed and yield
Extreme robustness

Extreme fidelity - A New Standard
Using a lacI-based method modified from previous studies, the error rate of Phusion 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 polymerase has the highest processivity of all thermostable DNA polymerases tested.

Table 1. The relative processivity values of Phusion and other DNA polymerases.
Polymerase	Relative processivity value
Phusion	10
Thermococcus kodakaraensis	8
Pyrococcus furiosus	1
Thermus aquaticus	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 dNTPs. 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 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 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.

Cycling protocols and enzyme amounts:
	Pyrococcus furiosus	modified P.furiosus	Phusion
Enzyme amount	5 U / 50 µl	2.5 U / 50 µl	1 U / 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
Cycle number	30	30	30

*Extension times: a) 1 min, b) 1 min 30 s, c) 3 min 50 s and d) 7 min 40 s.

Extreme robustness - minimize reaction failures
The random amplification of complex genomic Thermus species library illustrates the robust performance of Phusion polymerase. Phusion 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.

Phusion
		• 0.4 U / 20 µl rxn • 3 min extension time • 15 of 16 clones amplified
*Pyrococcus furiosus*
		• 1.0 U / 20 µl rxn • 10 min extension time • 9 of 16 clones amplified
*Thermus aquaticus*
		• 0.5 U / 20 µl rxn • 3 min extension time • 10 of 16 clones amplified

Figure 5 (on the right). 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. All 16 amplicons were amplified in Phusion HF buffer using the same reaction conditions according to supplier’s instruction.

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