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 [1] compared to the fidelity of Taq. Fidelity of Taq is represented here as 1x.
Phusion DNA Polymerases
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 of various inhibitors, allowing for robust amplification of PCR products with minimal optimization.
Featured video
Technical Features
- 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
Customer stories
"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 translating a DNA fragment into a protein.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 [1], is approximately 50-fold lower than that of Taq DNA polymerase and 6-fold lower than that of Pfu DNA polymerase (see Figure 1).
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 product 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 Thermo Scientific 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.
Select the best Phusion DNA polymerase product
Phusion High Fidelity DNA Polymerases are available in a variety of formats enabling high-fidelity PCR for even specialized applications. Phusion High-Fidelity DNA polymerase offers very high fidelity, enhanced thermostability, robustness and high tolerance for inhibitors in PCR. Use the table below find the appropriate Phusion format for your application.
| What feature do you need for your high-fidelity PCR application? | Recommended products and formats |
|---|---|
| High thermostability, robust PCR and inhibitor tolerance | Phusion High-Fidelity DNA Polymerase |
| All the products below include all of the advantages of the standard Phusion High-Fidelity DNA Polymerase products listed above. | |
|
Phusion Hot Start II DNA Polymerase and PCR Master Mix |
|
Phusion Flash High-Fidelity PCR Master Mix |
|
Phusion Site-Directed Mutagenesis Kit |
|
Phusion U Hot Start DNA Polymerase and PCR Master Mix |
|
Phusion U Multiplex PCR Master Mix |
|
Specialized Phusion buffers
|
| All standalone enzymes are supplied with HF buffer for highest fidelity and GC buffer for amplification of difficult/GC rich targets. All master mix formats provide a convenient 2X that includes all reaction components except the template and primers. |
|
Resources
Support
For Research Use Only. Not for use in diagnostic procedures.