Advantages of Phusion® DNA Polymerases

Accuracy - The highest fidelity of any available thermostable polymerase
Speed - Increased processivity allows shorter reaction times (extension 15-30 s/kb)
Robustness - Fewer reaction failures and minimal optimization
High yields - Increase product yields with minimal enzyme amounts
Specificity - Hot start modification reduces non-specific amplification and primer degradation

Extreme accuracy

Phusion DNA Polymerases have extremely low error rate, thus setting a new standard for high-fidelity PCR. The error rate, determined by a modified lacI-based method1, is 4.4 x10^-7 in HF Buffer. It is approximately 50-fold lower than that of Thermus aquaticus DNA polymerase and 6-fold lower than that of Pyrococcus furiosus DNA polymerase. Due to their low error rate, Phusion DNA Polymerases are ideally suited for cloning. Phusion DNA Polymerases produce blunt end PCR products.

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.

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Extreme speed

The Phusion DNA Polymerase has the highest processivity of all thermostable DNA polymerases tested. The increased processivity allows shorter reaction times (extension 15-30 s/kb)

Table 1. The relative processivity values of Phusion DNA Polymerase and other DNA polymerases.

Polymerase	Relative processivity value
Phusion DNA Polymerase	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® 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.

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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.

Phusion DNA Polymerase
		• 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 4 (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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Extreme 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.

Cycling protocols and enzyme amounts:
	Pyrococcus furiosus	modified P.furiosus	Phusion DNA Polymerase
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.

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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.

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