Artistic rendering of Phusion Plus Polymerase synthesizing DNA

Higher success, simpler setup, trusted PCR enzyme

Thermo Scientific Phusion Plus DNA Polymerase is the latest addition to the Phusion high-fidelity DNA polymerase family, which has been referenced in thousands of research publications. As a hot-start, proofreading PCR enzyme, Phusion Plus DNA polymerase enables generation of PCR amplicons with very 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.

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Highlights

  • 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—efficient amplification of GC-rich sequences and inhibitor-containing DNA; fast extension at 15–30 sec/kb
  • Fewer pipetting steps—master mix formats available with and without direct gel-loading dyes


Improved for more convenience and success to your PCR

Video: PCR with higher success, simpler success, and a trusted enzyme

Watch the video on five improved benefits of Phusion Plus DNA Polymerase over its predecessors.

Supporting data for Phusion Plus DNA Polymerase

Accuracy of PCR sequences

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

Bar graph of fidelity values 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.

Technical note: Measuring fidelity of Phusion Plus DNA Polymerase

Convenience of universal primer annealing

Due to its unique buffer formulation, Phusion Plus DNA Polymerase no longer requires a primer melting temperature (Tm ) calculator to determine the annealing temperatures. All targets can be amplified using a universal annealing temperature of 60°C regardless of the calculated primer Tms .The enzyme also works with the calculated annealing temperatures following the original Phusion protocol.

PCR gel images of a universal annealing temperature vs. calculated annealing temperatures

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.

PCR comparing co-cycling vs. individual cycling of different targets

Figure 3. 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.

PCR gel images of 0.3 to 7.5 kb amplicons using different DNA polymerases
Figure 4. 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 hr, enabling high-throughput PCR with a robotic or liquid-handling system.

Figure 5. 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 detect targets from as little as 0.08 ng of human genomic gDNA.

Gel images showing PCR sensitivity using different DNA polymerases
Figure 6. 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.

Gel images showing enzyme inhibitor tolerance using different DNA polymerases
Figure 7. 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.

PCR gel images of five GC-rich targets

Figure 8. 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 amplicon lengths

High processivity of Phusion Plus DNA NA Polymerase enables amplification of long DNA fragments—up to 10 kb from human gDNA and 20 kb from lambda DNA. Its cycling time is considerably short due to a fast extension rate at 15–30 sec/kb.

PCR gel images of 9 kb and 18 kb targets

Figure 9. 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.

Comparison of Phusion Plus, Phusion, and Phusion Hot-start II DNA polymerases

The following table compares technical features of Phusion Plus DNA polymerase and its predecessors. Its improved features are shaded in blue.

 Phusion Plus DNA PolymerasePhusion High-Fidelity DNA PolymerasePhusion Hot-Start II DNA Polymerase

Fidelity for sequence accuracy

(vs. Taq enzyme)

>100x52x52x
Use of Tm calculatorNot requiredRequiredRequired
Hot-start modification for specificityYesNoYes
PCR sensitivity+++++++

Amplification length

(human gDNA / lambda DNA)

Up to 10 kb / 20 kbUp to 7.5 kb / 20 kbUp to 7.5 kb / 20 kb

GC-rich amplification

+++++++
Inhibitor tolerance+++++++
Standalone enzyme
Master mix

*Contains green tracking dyes and density reagent for direct gel-loading of PCR products
Performance comparison between Phusion Plus and Phusion DNA polymerases

For more Phusion formats, please visit their selection table

Ordering information for Phusion Plus DNA Polymerase

To order the original Phusion and related products, please visit their ordering table

Resources

Find videos, webinars, articles, and tools in our molecular biology resource library

Manuals / protocols

Flyers / brochures

Technical notes

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