Thermo Scientific Phusion DNA Polymerases are high fidelity polymerases created by fusing a dsDNA-binding domain with a Pyrococcus polymerase-like proofreading enzyme. The proofreading capabilities of Phusion DNA Polymerases result in exceptionally low error rates, rapid extension times, and tolerance to inhibitors. Phusion DNA Polymerases come in various formats and are particularly well-suited for applications that require high accuracy, such as cloning, sequencing, and mutagenesis. Thermo Scientific Phusion Plus DNA Polymerase is the latest addition to the Phusion high-fidelity DNA polymerase family, which has higher fidelity compared to other Phusion family products and it also simplifies the primer annealing step with a universal annealing feature, helping you to skip calculating primer annealing temperatures.


Phusion DNA Polymerases: Selection guide

Phusion high-fidelity DNA polymerases are available in a variety of formats. The table below compares the various formats of Phusion DNA polymerases.

 

product package with three tubes of Phusion Plus DNA polymerase reagents

Phusion Plus DNA Polymerase

Five tubes of Phusion High Fidelity DNA polymerase reagents

Phusion High-Fidelity DNA Polymerase

Five tubes of Phusion Hot Start II DNA polymerase reagents

Phusion Hot Start II DNA Polymerase

single tube with Phusion Flash High-Fidelity PCR Master Mix colorless reagent

Phusion Flash High-Fidelity PCR Master Mix

four tubes of Phusion U Hot Start DNA reagents placed in a row

Phusion U Hot Start DNA Polymerase

four tubes of Phusion U Multiplex PCR Master Mix reagents placed in a row

Phusion U Multiplex PCR Master Mix

Fidelity (vs. Taq enzyme)>100x 52x52x25x25x25x
Tm calculation requiredNoYesYesYesYesYes
Enhanced specificity (hot-start version)YesNoYesYesYesYes
Amplification lengthUp to 20 kbUp to 20 kbUp to 20 kbUp to 20 kb Up to 20 kbUp to 2.5 kb
Speed15–30 sec/kb15–30 sec/kb15–30 sec/kb<15 sec/kb15–30 sec/kb15–30 sec/kb
GC-rich formatYesYesYesNoYesNo
dUTP incorporationNoNoNoNoYesYes
Designed for multiplex PCRNoNoNoNoNoYes, up to 20 targets
Stand-alone enzyme format*

Colorless 
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Colorless
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Green**
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Colorless
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Green**
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N/A

Colorless

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Green**
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N/A
2x master mix format*

Colorless 
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Green**
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Colorless with HF Buffer
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Colorless with GC Buffer
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Colorless
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Green**
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Colorless
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Colorless
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Colorless
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Green**
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*All stand-alone enzymes are supplied with their reaction buffers. The 2x master mix formats include all necessary PCR components except the template and primers.
**Contains green loading dyes and a density reagent for direct gel loading of PCR products.

Phusion Plus DNA Polymerase

Phusion Plus DNA Polymerase is the latest generation of Phusion DNA polymerase that is widely cited in peer-reviewed publications. As a hot-start, proofreading PCR enzyme, Phusion Plus DNA polymerase enables generation of PCR amplicons with high sequence accuracy, sensitivity, and specificity. It also simplifies the primer annealing step with a universal annealing feature, allowing you to skip calculating primer annealing temperatures and use 60°C annealing temperature for many of the primers.

Benefits of Phusion Plus DNA Polymerase

  • High sequence accuracy—PCR with >100x fidelity of Taq enzyme
  • Simplified reaction setup—Calculation of primer annealing temperatures no longer required due to the unique buffer formulation
  • Enhanced specificity and benchtop stability—Hot-start modification improves PCR specificity and room temperature stability of assembled reactions
  • Robust PCR—Fast extension rates at 15–30 sec/kb; efficient amplification of GC-rich sequences and inhibitor-containing DNA 
  • Fewer pipetting steps—Master mix formats available with and without direct gel-loading dyes 

Benchmarking data for Phusion Plus DNA Polymerase

Sequence accuracy

As a proofreading enzyme with very high fidelity, Phusion Plus DNA Polymerase generates amplicons with very few errors, helping you with PCR sequence accuracy.

Read technical note:  Measuring fidelity of Phusion Plus DNA Polymerase

Bar graph of accuracy of high-fidelity PCR enzymes

Figure 1. Fidelity of high-fidelity polymerases relative to Taq DNA polymerase. Error rates were determined by next-generation sequencing using molecular barcodes, then normalized to that of Taq DNA polymerase.

Convenience of universal primer annealing

Skip calculating primer melting temperature (Tm). Due to Phusion Plus DNA Polymerase's unique buffer formulation, all targets can be amplified using a universal annealing temperature of 60°C regardless of the calculated primer Tms. Phusion Plus DNA Polymerase also works with the calculated annealing temperatures following the original Phusion protocol.

Composite of two gel images: each image has 12 sample lanes flanked by ladder bands
Figure 2. PCR cycling under two annealing conditions. 12 targets with varying calculated annealing temperatures (indicated above each lane) were amplified from 50 ng of human genomic DNA (gDNA), following a universal annealing temperature of 60°C (left), or the annealing temperatures calculated with the Tm calculator (right). The molecular weight marker is Thermo Scientific ZipRuler Express Ladder DNA 2.

The universal annealing feature of Phusion Plus DNA Polymerase also allows a universal cycling protocol, helping you to circumvent multiple PCR runs and save time. One annealing temperature (60°C) and one extension time based on the longest amplicon can be used for targets of different lengths—i.e., co-cycling different targets on the same block—without compromising PCR yields and specificity. 

Schematic diagram comparing the results of one cycling different cycling protocols

Figure 3. Single PCR runs help save time.

Figure 4. Different PCR targets can be co-cycled using Phusion Plus DNA polymerase. Five targets of different lengths were amplified from human gDNA using a universal cycling protocol for all targets (up to 7.5 kb), with the extension time of the longest amplicon (3 min 45 sec for 7.5 kb) (left), or following separate cycling protocols with a different extension time calculated for each target (9 sec for 0.3 kb, 21 sec for 0.7 kb, 60 sec for 2 kb, 2 min for 3.9 kb, 3 min 45 sec for 7.5 kb) (right). The molecular weight marker is ZipRuler Express DNA Ladder 2.
Yields and specificity of PCR amplicons

Phusion Plus DNA Polymerase improves PCR specificity and yields since its affibody molecule-mediated hot-start modification prevents enzyme activity and primer degradation during reaction setup.

Figure 5. High yields and specificity using Phusion Plus DNA Polymerase. 0.3–7.5 kb DNA targets were amplified from 100 ng of human gDNA using various DNA polymerases according to the manufacturer recommendations. The molecular weight marker is Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.
Stability of assembled reactions

Due to its stringent hot-start modification, Phusion Plus DNA Polymerase provides stability of assembled reactions at room temperature for up to 24 hours, enabling high-throughput PCR with a robotic or liquid-handling system.

Figure 6. Benchtop stability of Phusion Plus DNA Polymerase. A 0.5 kb target was amplified from 50 ng of human gDNA using various hot-start DNA polymerases according to the manufacturer recommendations. Assembled PCR reactions were loaded immediately onto a thermal cycler (0 hr) or set at room temperature for 24 hr before cycling (24 hr). The molecular weight marker is GeneRuler 1 kb Plus DNA Ladder.
Sensitivity in target detection

Phusion Plus DNA Polymerase can amplify targets from as little as 0.08 ng of human genomic gDNA.

Figure 7. High sensitivity of Phusion Plus DNA Polymerase. A 0.5 kb target was amplified from different amounts of human gDNA using various DNA polymerases according to the manufacturer recommendations. The molecular weight marker is GeneRuler 1 kb Plus DNA Ladder.
Tolerance to PCR inhibitors

By combining its fusion protein technology with buffer reformulation, Phusion Plus DNA Polymerase can better tolerate inhibitors from plants (e.g., xylan), soil (e.g., humic acid), and blood (e.g., hemin) during PCR.

Figure 8. High inhibitor tolerance of Phusion Plus DNA Polymerase. A 2 kb target was amplified from 50 ng of human gDNA using various DNA polymerases according to the manufacturer recommendations. The reaction mixtures contained at a final concentration of 1: no inhibitor, 2: humic acid (0.5 µg/mL), 3: hemin (2.5 µM), or 4: xylan (250 µg/mL). The molecular weight marker is GeneRuler 1 kb Plus DNA Ladder.

Amplification of GC-rich sequences

Phusion Plus DNA Polymerase includes Phusion GC Enhancer in the package for more efficient amplification of sequences with >65% GC content.

Figure 9. Efficient GC-rich amplification of Phusion Plus DNA Polymerase. Five targets (0.78 kb, 0.74 kb, 0.72 kb, 0.66 kb, and 0.55 kb) with high GC content (their percentages indicated) were amplified from 50 ng of human gDNA using various DNA polymerases according to the manufacturer recommendations for GC-rich PCR. The molecular weight marker is ZipRuler Express Ladder DNA 2.
Range of amplicons

High processivity of Phusion Plus DNA Polymerase enables amplification of long DNA fragments—up to 10 kb from human gDNA and 20 kb from lambda DNA.

Composite of two gels: each gel has six lanes and is flanked on both sides by DNA ladders
Figure 10. Amplification ranges of Phusion Plus DNA polymerase. A 9.1 kb target of human gDNA (left) and an 18 kb target of lambda DNA (right) were successfully amplified from two template amounts using Phusion Plus DNA Polymerase. The molecular weight marker is GeneRuler 1 kb Plus DNA Ladder.

Resources for Phusion Plus DNA Polymerase

Manuals/Protocols

Flyers/Brochures

Technical notes

Additional resources

Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.