Preferred high-fidelity DNA polymerases for accuracy, speed, and value
Since their introduction, Thermo Scientific Phusion High-Fidelity DNA Polymerases have been referenced in thousands of publications for high-performance PCR and have become the choice for a multitude of demanding applications ranging from reconstruction, design and massively-parallel, high-throughput sequencing of whole genomes. The Phusion family of products includes over a dozen varied formats enabling high-fidelity PCR for even specialized applications.
Why use Phusion High-Fidelity DNA polymerase?
Phusion High-Fidelity DNA Polymerase is comprised of a DNA-binding domain fused to a Pyrococcus-like proofreading polymerase. As a result of this unique functional pairing, Phusion DNA Polymerases are capable of generating PCR products with very high accuracy and speed. In addition, Phusion DNA Polymerases are tolerant to various inhibitors, allowing for robust amplification of PCR products with minimal optimization.
- High fidelity—52x more accurate than Taq, 6x more accurate than Pfu
- Enhanced robustness—fewer reaction failures and minimal optimization
- Improved yields—high product yields with minimal enzyme amounts (0.5–1 U/50 μL reaction)
- High speed—increased processivity allows shorter reaction times (extension 15–30 s/kb)
- Enhanced specificity—unique hot start technology with no reactivation time reduces nonspecific amplification and primer degradation
- Simplified workflows—Phusion Green DNA Polymerases allow direct loading of PCR products onto gels
"After realizing that we could get the same number of cycles in roughly a quarter of the time (and at only slightly higher per unit cost), we changed exclusively to Phusion."
—Matt W. Ford, PhD student, Department of Biological Sciences, Idaho State University, USA.
"Different thermostable DNA polymerases were tested, but only Phusion polymerase, a Pyrococcus DNA polymerase-like enzyme fused with a double-stranded DNA-binding domain, had sufficient processivity."
—Hass M et al. (2008) J. Virol. 82:10207-10217.
"Earlier attempts with Turbo-Pfu polymerase were not successful. However, when using the highly processive Phusion polymerase instead, the usage of vector pHWSccdB led to positive clones."
—Stech J et al. (2008)
Nucleic Acids Research, 36:e139.
Features of Phusion DNA polymerases
It is crucial to maintain the accurate DNA sequence during amplification, especially in applications such as cloning, site-directed mutagenesis, or transcription and translation of a PCR fragment. One incorrectly incorporated nucleotide may change the respective codon and result in the addition of an incorrect amino acid during translation. In turn, this can affect folding and functional properties of the protein. On the other hand, deletion of a single nucleotide completely destroys the correct reading frame.
Phusion DNA Polymerases exhibit very low error rates and as a result are ideal for accuracy in high-fidelity PCR applications such as cloning subcloning, sample preparation for NGS, and site-directed mutagenesis. The error rate, determined by a modified lacI-based method , is approximately 50-fold lower than that of Taq DNA polymerase and 6-fold lower than that of Pfu DNA polymerase (see Figure 1).
Figure 1. The bar graph represents relative fidelity values of Phusion DNA Polymerase and high-fidelity polymerases from other suppliers, obtained by the modified lacI-based method  compared to the Taq DNA polymerase's fidelity. Fidelity of Taq DNA polymerase is represented here as 1x.
Due to the unique structure of the enzyme, Phusion DNA polymerases are also highly efficient. When compared to conventional polymerases, significantly fewer units of the enzyme are required for any PCR reaction. Speed and efficiency result in high yields and very high specificity. Also, these polymerases are highly robust which minimizes the need for reaction optimization. Compared with leading competitor high-fidelity polymerases, Phusion Hot Start II DNA Polymerase shows greater sensitivity in PCR with specific amplification of target fragments from human genomic DNA (Figure 2).
Figure 2. High sensitivity and specific PCR amplification. Phusion Hot Start II Green Master Mix (A) and proofreading hot-start DNA polymerases from other suppliers (B) Merck KOD Hot Start, (C) Agilent Pfu Ultra II HS, (D) KAPA Biosystems KAPA HiFi HS, and (E) Takara PrimeSTAR MAX were used to amplify a 2 kb fragment from human genomic DNA. Phusion Hot Start II DNA Polymerase provided high yields of specific products, whereas other enzymes delivered low yields, with some also amplifying nonspecific products.
Phusion DNA polymerases incorporate more nucleotides per binding event as compared to other polymerases. This high processivity allows extremely short extension times and consequently reduced protocol times. Rapid protocol times can be achieved with Phusion Flash High-Fidelity PCR Master Mix, a product developed especially for fast PCR. Phusion Flash High-Fidelity PCR master mix outperforms other competitor products yielding specific products with extension times of as little as 10s/kb (Figure 3).
Figure 3. High speed and yields with Phusion Flash High-Fidelity PCR Master Mix. A 1.5 kb human cathepsin K gene was amplified with (A) Phusion Flash High-Fidelity PCR Master Mix and polymerases from other suppliers including (B) Agilent Pfu Ultra II Fusion and (C) Qiagen Fast Cycling 2X Mix using varying extension times (10–60 seconds) according the manufacturer’s recommendations. Only Phusion Flash High-Fidelity PCR Master Mix was able to amplify the 1.5 kb gene with very short extension times of 10 and 20 seconds. It also produced superior yields of specific product compared to the other enzymes tested.
PCR inhibition is a common problem in DNA amplification failure. Most inhibitors interfere with target DNA or the DNA polymerase itself, blocking enzymatic activity. Phusion High-Fidelity and Phusion Hot Start II DNA polymerases have greater resistance to PCR inhibitors than other leading competitor high-fidelity polymerases.
Figure 4. A 2 kb fragment from 50 ng human genomic DNA was amplified by various DNA polymerases according to recommendations provided by the manufacturer with addition of common PCR inhibitors in reaction mixture (1—no inhibitor, 2—humic acid (0.125 µg/mL), 3—hemin (0.625 µM), or 4—bile salt (1 mg/mL)). The molecular weight marker is ZipRuler Express DNA Ladder 2.
One of the major benefits of a hot start polymerase is its stability at room temperature. Not only does this reduce nonspecific amplification, but it also allows for a more convenient reaction setup and use in high-throughput applications. Phusion Hot Start II polymerases are stable up to 24 hours at room temperature compared to other leading PCR enzymes.
Figure 5. A 0.5 kb fragment from 50 ng of human genomic DNA was amplified by various hot-start DNA polymerases according to recommendations provided by the manufacturer. PCR reactions were set up and incubated at room temperature for 0h and 24h before loading in the thermal cycler. The molecular weight marker is ZipRuler Express DNA Ladder 2.
Amplification of a broad range of amplicon lengths is an important consideration in choosing a high-fidelity PCR enzyme. Phusion High-Fidelity DNA polymerases are able to amplify targets of various amplicon lengths more consistently and robustly than other leading enzymes.
Figure 6. Robust amplification across broad range of amplicon lengths. Phusion High Fidelity DNA Polymerase amplifies various length fragments with high specificity and yield. Four DNA fragments from 300 bp to 7.5 kb were amplified from 100 ng of human genomic DNA by various DNA polymerases according to recommendations provided by the manufacturer. The molecular weight marker is ZipRuler Express DNA Ladder 2.
Select a Phusion DNA polymerase best suited for your application
Phusion high-fidelity DNA polymerases are available in a variety of formats. Please reference the table below to find an appropriate Phusion format for your application.
|Phusion High-Fidelity DNA Polymerase||Phusion Hot Start II DNA Polymerase||Phusion Flash High-Fidelity PCR Master Mix||Phusion U Hot Start DNA Polymerase||Phusion U Multiplex PCR Master Mix|
|Fidelity (vs. Taq enzyme)||52x||52x||25x||25x||25x|
|Enhanced specificity (hot-start version)||No||Yes||Yes||Yes||Yes|
|Amplification length||Up to 20 kb||Up to 20 kb||Up to 20 kb||Up to 20 kb||Up to 2.5 kb|
|Speed||15–30 sec/kb||15–30 sec/kb||<15 sec/kb||15–30 sec/kb||15–30 sec/kb|
|Compatible with dUTP||No||No||No||Yes||Yes|
|Designed for multiplex PCR||No||No||No||No||Yes, up to 20 targets|
|Stand-alone enzyme format*|
|2x master mix format*|
* All stand-alone enzymes are supplied with HF buffer for highest fidelity and GC buffer for amplification of GC-rich targets. The 2x master mix formats include all necessary PCR components except the template and primers.
** Contains an inert green dye for direct gel loading of PCR products
Ordering information for Phusion high-fidelity DNA polymerases
Do you need a DNA polymerase with higher fidelity and/or performance than Phusion enzymes? Discover Invitrogen Platinum SuperFi II DNA Polymerase with >300x Taq fidelity and a simplified PCR protocol.
For Research Use Only. Not for use in diagnostic procedures.