 | Since their introduction in 2003, Thermo Scientific Phusion High-Fidelity DNA Polymerases have established a new standard for high-fidelity PCR. Phusion DNA Polymerases have proven first choice for several demanding PCR applications, including the creation of first functional synthetic genome. |
Overview
In Phusion High-Fidelity DNA Polymerases, a DNA binding domain is fused to a Pyrococcus-like proofreading polymerase. Due to this unique fusion technique, Phusion DNA Polymerases generate PCR products with accuracy and speed unattainable with a single enzyme, even on the most difficult templates. In addition, Phusion DNA Polymerases are tolerant of various PCR inhibitors. This allows robust amplification with minimal optimization. For hot-start PCR, Phusion Hot Start II High-Fidelity DNA Polymerase is an ideal choice allowing extreme specificity and improved robustness.
Customer Testimonials
"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
Extreme fidelity for demanding PCR
In many applications such as cloning, site-directed mutagenesis or translating a DNA fragment into a protein, it is crucial to maintain the accurate DNA sequence during amplification. One incorrectly incorporated nucleotide may change the respective codon and result in the addition of an incorrect amino acid during translation. This, in turn, 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 have extremely low error rates, thus setting a gold standard for Taq PCR. The error rate, determined by a modified lacI-based method1, is approximately 50-fold lower than that of DNA polymerase and 6-fold lower than that of Pfu DNA polymerase (see Chart 1).
Chart 1. Relative fidelity values of different DNA polymerases. Fidelity = 1 / error rate. The low error rate of Phusion DNA Polymerase was recently confirmed by studies using 454 sequencing2 and Illumina sequencing3. With 454 sequencing, Phusion DNA Polymerase was shown to have the lowest error rate compared to other DNA polymerases (see Table 1).
Table 1. Error rates determined by 454 sequencing for seven different DNA polymerases after PCR amplification of four different exons from the human TP53 oncogene, using a clonal TP53 plasmid as starting template.
aError rate, number of errors (miscalled bases, inserted or deleted bases) divided by total number of bases.bDot, three successive negative flows during 454 sequencing. Table reprinted with the permission from the corresponding author and the journal editor.
B.F. Frey & B. Suppmann (1995) Demonstration of the Expand PCR System's greater fidelity and higher yields with a lacI-based fidelity assay. Biochemica 2, 34-35.Vandenbroucke et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications. Biotechniques. 53:167-177.Kinde et al. (2011) Detection and quantification of rare mutations with massively parallel sequencing. PNAS. 108(23):9530–9535.
Increased processivity allows short reaction times (extension 15-30 s/kb)
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. Shortest protocol times can be achieved with Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix, a product developed especially for fast PCR.
Extreme speed and high yields with Phusion Flash. A 1.5 kb human cathepsin K gene was amplified with three different polymerases using varying extension times (10-60 s) with Piko® Thermal Cycler. Only Phusion Flash was able to amplify the 1.5 kb gene with extremely short extension times of 10 and 20 s. It also produced superior yields of specific product compared to other enzymes tested.
Fewer reaction failures and minimal optimization
Phusion DNA Polymerases are exceptionally tolerant of inhibitors and other challenging reaction conditions. This provides reliability by minimizing reaction failures, and even enables PCR from unpurified sample materials. Phusion DNA Polymerases are the key components in Thermo Scientific Direct PCR Kits for human specimens and blood samples.
 |
+ and - denote control reactions with and without purified DNA. |
PCR from various human-derived samples without DNA purification. Different samples were placed directly into 20 μl PCR reactions. A 237 bp DNA fragment was successfully amplified using the control primers included in the Phusion Human Specimen Direct PCR Kit. Reactions were run in Thermo Scientific UTW reaction vessels using Thermo Scientific Piko Thermal Cycler.
High product yields with minimal enzyme amounts
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 in minimal time. Also, these polymerases are highly robust which minimizes the need for reaction optimization.
Less enzyme - superior yield. A 3.8 kb fragment from human beta globin gene was amplified with threedifferent DNA polymerases. Phusion DNA Polymerase was able to amplify the 3.8 kb genomic fragment with a combined annealing and extensionstep of only 1 minute, thus being significantly faster than the two otherpolymerases tested. A single unit of Phusion DNA Polymerase produced higher yields than 2.5 or 5 units of the Pfu DNA polymerases.
Extreme specificity with the hot-start modification
The Affibody®-based inactivation method of the Thermo Scientific Phusion Hot Start II DNA Polymerase greatly increases the specificity of PCR amplification. Both the DNA polymerase and the proofreading activities of Phusion Hot Start II DNA Polymerase are inactivated at room temperature by a reversibly binding Affibody ligand. This prevents nonspecific extension of the DNA template as well as degradation of the PCR primers during reaction setup. With Phusion Hot Start II DNA Polymerase, the reaction setup can be done at room temperature, enabling its use in high-throughput robotics. In addition to improved specificity, Phusion Hot Start II DNA Polymerase delivers improved robustness, allowing fewer reaction failures and minimizing the need for optimization.
Phusion Hot Start II DNA Polymerase provides extreme specificity and abundant yields. Five proofreading hot start DNA polymerases from major suppliers were used to amplify 1.7-2.3 kb fragments from human genomic DNA. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered zero or low yields, with some also amplifying non-specific products.
Direct loading of PCR products on gels
Phusion Green format offers high convenience for the PCR setup and detection. This format is a combination of Phusion DNA Polymerase and 5X Phusion Green Buffer which includes a density reagent and two tracking dyes for direct loading of PCR products on gels. The Green Buffer does not interfere with the performance of Phusion DNA Polymerase and is compatible with downstream applications including DNA sequencing, ligation and restriction digestion.
 | 1X reaction mixture containing Phusion Green Buffer A – unseparated in a well B – blue and yellow dyes following electrophoresis |
Extreme fidelity for demanding PCR
In many applications such as cloning, site-directed mutagenesis or translating a DNA fragment into a protein, it is crucial to maintain the accurate DNA sequence during amplification. One incorrectly incorporated nucleotide may change the respective codon and result in the addition of an incorrect amino acid during translation. This, in turn, 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 have extremely low error rates, thus setting a gold standard for Taq PCR. The error rate, determined by a modified lacI-based method1, is approximately 50-fold lower than that of DNA polymerase and 6-fold lower than that of Pfu DNA polymerase (see Chart 1).
Chart 1. Relative fidelity values of different DNA polymerases. Fidelity = 1 / error rate. The low error rate of Phusion DNA Polymerase was recently confirmed by studies using 454 sequencing2 and Illumina sequencing3. With 454 sequencing, Phusion DNA Polymerase was shown to have the lowest error rate compared to other DNA polymerases (see Table 1).
Table 1. Error rates determined by 454 sequencing for seven different DNA polymerases after PCR amplification of four different exons from the human TP53 oncogene, using a clonal TP53 plasmid as starting template.
aError rate, number of errors (miscalled bases, inserted or deleted bases) divided by total number of bases.bDot, three successive negative flows during 454 sequencing. Table reprinted with the permission from the corresponding author and the journal editor.
B.F. Frey & B. Suppmann (1995) Demonstration of the Expand PCR System's greater fidelity and higher yields with a lacI-based fidelity assay. Biochemica 2, 34-35.Vandenbroucke et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications. Biotechniques. 53:167-177.Kinde et al. (2011) Detection and quantification of rare mutations with massively parallel sequencing. PNAS. 108(23):9530–9535.
Increased processivity allows short reaction times (extension 15-30 s/kb)
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. Shortest protocol times can be achieved with Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix, a product developed especially for fast PCR.
Extreme speed and high yields with Phusion Flash. A 1.5 kb human cathepsin K gene was amplified with three different polymerases using varying extension times (10-60 s) with Piko® Thermal Cycler. Only Phusion Flash was able to amplify the 1.5 kb gene with extremely short extension times of 10 and 20 s. It also produced superior yields of specific product compared to other enzymes tested.
Fewer reaction failures and minimal optimization
Phusion DNA Polymerases are exceptionally tolerant of inhibitors and other challenging reaction conditions. This provides reliability by minimizing reaction failures, and even enables PCR from unpurified sample materials. Phusion DNA Polymerases are the key components in Thermo Scientific Direct PCR Kits for human specimens and blood samples.
|
+ and - denote control reactions with and without purified DNA. |
PCR from various human-derived samples without DNA purification. Different samples were placed directly into 20 μl PCR reactions. A 237 bp DNA fragment was successfully amplified using the control primers included in the Phusion Human Specimen Direct PCR Kit. Reactions were run in Thermo Scientific UTW reaction vessels using Thermo Scientific Piko Thermal Cycler.
High product yields with minimal enzyme amounts
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 in minimal time. Also, these polymerases are highly robust which minimizes the need for reaction optimization.
Less enzyme - superior yield. A 3.8 kb fragment from human beta globin gene was amplified with threedifferent DNA polymerases. Phusion DNA Polymerase was able to amplify the 3.8 kb genomic fragment with a combined annealing and extensionstep of only 1 minute, thus being significantly faster than the two otherpolymerases tested. A single unit of Phusion DNA Polymerase produced higher yields than 2.5 or 5 units of the Pfu DNA polymerases.
Extreme specificity with the hot-start modification
The Affibody®-based inactivation method of the Thermo Scientific Phusion Hot Start II DNA Polymerase greatly increases the specificity of PCR amplification. Both the DNA polymerase and the proofreading activities of Phusion Hot Start II DNA Polymerase are inactivated at room temperature by a reversibly binding Affibody ligand. This prevents nonspecific extension of the DNA template as well as degradation of the PCR primers during reaction setup. With Phusion Hot Start II DNA Polymerase, the reaction setup can be done at room temperature, enabling its use in high-throughput robotics. In addition to improved specificity, Phusion Hot Start II DNA Polymerase delivers improved robustness, allowing fewer reaction failures and minimizing the need for optimization.
Phusion Hot Start II DNA Polymerase provides extreme specificity and abundant yields. Five proofreading hot start DNA polymerases from major suppliers were used to amplify 1.7-2.3 kb fragments from human genomic DNA. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered zero or low yields, with some also amplifying non-specific products.
Direct loading of PCR products on gels
Phusion Green format offers high convenience for the PCR setup and detection. This format is a combination of Phusion DNA Polymerase and 5X Phusion Green Buffer which includes a density reagent and two tracking dyes for direct loading of PCR products on gels. The Green Buffer does not interfere with the performance of Phusion DNA Polymerase and is compatible with downstream applications including DNA sequencing, ligation and restriction digestion.
| 1X reaction mixture containing Phusion Green Buffer A – unseparated in a well B – blue and yellow dyes following electrophoresis |
Applications of Phusion DNA Polymerases
High-fidelity PCR
Due to its very high accuracy, Phusion DNA Polymerase is considered as a golden standard for high-fidelity PCR. Phusion is 52x more accurate than Taq and 6x more accurate than Pfu DNA polymerase and is therefore used for all high fidelity PCR applications, such as cloning or template generation for sequencing.
Amplification of long amplicons
Phusion DNA Polymerases are the ideal choice for amplification of long templates up to 20 kb. Due to the high processivity, and short cycling times, high yields of long amplicons are produced in a fraction of the time other polymerases need.
Fast PCR
Increased processivity of Phusion DNA Polymerases allows shorter extension times and therefore faster PCR protocols. To further reduce protocol time, we recommend the combination of Phusion Flash High Fidelity PCR Master Mix, Piko Thermal Cycler and UTW PCR plastics.
Hot-Start PCR
Phusion Hot Start II High-Fidelity DNA Polymerase combines the Phusion DNA Polymerase and a reversibly bound, specific Affibody ligand. The ligand inhibits the 5‘- 3' polymerase activity and 3'-5' exonuclease activity (proofreading) at room temperature, but renders the polymerase fully active at polymerization temperatures.
DHPLC and Microarray applications
Detergent-free reaction buffers are available for Phusion DNA Polymerase. PCR products used for DHPLC analysis are recommended to be free of detergents. Detergents may also cause foaming when PCR products are spotted on microarray slides.
Amplification of difficult (GC-rich) templates
Specially optimized buffer system in Phusion High-Fidelity PCR Master Mix with GC Buffer enables polymerase to overcome difficulties in amplifying GC-rich templates.
High-throughput PCR
Phusion Hot Start II High-Fidelity PCR Master Mixes are suitable for robotic applications where room temperature reaction setup is required. The speed of the polymerase facilitates running fast protocols, which translates to more runs per day.
Mutagenesis
Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid DNA.
Multiplex PCR
High performance Phusion U Multiplex PCR Master Mix enables fast and reliable amplification of multiple targets from a broad range of DNA templates. It is possible to perform simultaneous amplification of over 20 of targets with high yields and specificity.
Amplification of dUTP-containing templates and dUTP incorporation
One of important limitations of proofreding DNA polymerases is their inability to amplify dUTP-containing templates and incorporate dUTP. Phusion U DNA polymerase carries mutation in the dUTP binding pocket to overcome these limitations. It can therefore be used in applications such as amplification of bisulfite-converted DNA or carry-over contamination control.
Phusion DNA Polymerase Products
Phusion High-Fidelity DNA Polymerase
Phusion High-Fidelity DNA Polymerase is a thermostable DNA polymerases with extremely high accuracy and performance that no other enzyme can match.
Phusion Green High-Fidelity DNA Polymerase
Phusion High-Fidelity DNA Polymerases supplied with the convenient Green Buffer for direct loading of PCR products on gels.
Phusion Flash High-Fidelity PCR Master Mix
Phusion Flash High-Fidelity PCR Master Mix is a 2x master mix enabling the use of extremely short PCR protocols without compromising either the fidelity or the yield in the reaction.
Phusion Hot Start II DNA Polymerase and PCR Master Mix
Phusion Hot Start II High-Fidelity DNA Polymerase offers extreme specificity and improved performance. The 2x master mix contains Phusion Hot Start II DNA Polymerase, nucleotides and optimized reaction buffer.
Phusion Green Hot Start II High-Fidelity DNA Polymerase and PCR Master Mix
Phusion Hot Start II High-Fidelity DNA Polymerase and PCR Master Mix supplied with the convenient Green Buffer for direct load on gels.
Phusion RT-PCR Kit
Phusion RT-PCR Kit is a complete two-step kit designed for high-fidelity RT-PCR. The kit contains the full set of reagents required for performing cDNA synthesis and PCR in two steps.
Phusion Site-Directed Mutagenesis Kit
The Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions or deletions in any type of plasmid DNA.
Phusion U Multiplex PCR Master Mix
High performance Phusion U Multiplex PCR Master Mix enables simultaneous amplification of over 20 targets up to 2.5 kb. It is based on Phusion U Hot Start DNA polymerases ensuring high yields of specific products.
Phusion U Hot Start DNA Polymerase and PCR Master Mix
Phusion U Hot Start DNA Polymerase is a proofreading enzyme with a proprietary mutation in the dUTP binding pocket which allows amplification of dUTP-containing templates and dUTP incorporation. The 2x master mix contains Phusion U Hot Start DNA Polymerase, nucleotides and optimized reaction buffer.
Phusion Buffers
Optimized buffers for Phusion DNA Polymerases. Also available as detergent-free options.