What is fusion DNA polymerase
Phusion High Fidelity DNA Polymerase
The Phusion® High Fidelity DNA Polymerase is an artificially produced "chimera" from a Pyrococcus-like polymerase and an additionally inserted dsDNA-binding protein domain. Thanks to this ssO7d domain, the processivity of the phusion polymerase - i.e. the "dwell time" per binding event - is dramatically increased compared to other enzymes: it "rushes" along the template DNA strand and the new DNA is synthesized in a fraction of the time. The Phusion Polymerase is therefore particularly fast and well suited for longer amplicons and for high accuracy!
- High accuracy: 52 times more accurate than Taq
- High speed: only 15-30 seconds / kb
- Long amplificates with high yields, even with difficult templates
- Phusion® Hot Start Flex variant: Aptamer technology for increased flexibility, specificity and yields without any heat inactivation
Fig .: Phusion DNA polymerase
The inserted double-strand binding "enhancer" domain (orange) enables extreme processivity, accuracy and reliability. Also available as a Phusion® Hot Start Flex variant: an aptamer technology enables increased flexibility, specificity and yields without any heat activation.
Fig .: A 3.8 kb fragment was amplified from 50 ng genomic Jurkat DNA with various polymerases according to the manufacturer's instructions. The length of the extension step is given in minutes. Ladder L is the 1 kb DNA ladder # N3232.
NEB's free Tm calculator:
Here you enter via "Copy-Paste"
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NEB PCR Polymerase you are using - done!
The program determines the optimal annealing temperature for perfect PCR results!
For all applications in which a correct DNA sequence is necessary (e.g. DNA cloning, protein expression, reporter gene assays) you need a polymerase with 3 '-> 5' exonuclease activity (called "proof-reading") for DNA amplification using PCR. This exonuclease "controls" every incorporation of a new dNTP during DNA synthesis and immediately "cuts out" an incorrectly incorporated base at the 3 'end. This enables an almost flawless synthesis. Well-known polymerases with proof-reading activity are for example NEBs Vent® and DeepVent® polymerase, Pyrocossus furiosus (Pfu) polymerase and other archaebacterial DNA polymerases as well as Phusion® DNA polymerase. The accuracy of the proof reading, i.e. the activity of the 3 ’-> 5’ exonuclease, differs from polymerase to polymerase and can be quantitatively measured and compared in so-called LacI assays.
Thermostable (PCR-capable) polymerases are usually inherently not particularly processive. This means that when the polymerase binds to the template DNA, the DNA strand to be synthesized is only lengthened by a few bases before the polymase dissociates and has to bind again. Thus, multiple binding events are necessary even for an extension of only short DNA sequences. The process of continuous “turn and go” is therefore the rate-determining and limiting step of the entire reaction.
Due to the “dissociative” character of the polymerases, the cycler / extension times of approx. 1 min per kilobase known from practice also result for Taq DNA polymerase. In the case of Pfu and other enzymes with proof-reading activity, even up to 2 minutes per kilobase are necessary for a sufficient amplification yield.
Phusion® DNA polymerase, on the other hand, only needs 15-30 seconds per kilobase for excellent yields!
Focus on difficult templates
DNA with special secondary structures, GC-rich or loop sequences is often very difficult or impossible to amplify: the polymerase can actually only bind again with great difficulty or not at all in these “difficult” sequences. An amplification of this section is therefore often not available. Thanks to its improved processivity, Phusion® Polymerase is also ideally suited for difficult and long amplicons. By choosing the enclosed GC buffer, secondary structures of the template DNA are well resolved and made accessible for PCR. Long amplicons, e.g. up to 20 kb on human gDNA, can be amplified very well with the Phusion® polymerase.
Example: A 3.8 kb fragment was amplified from Jurkat gDNA with different polymerases according to the manufacturer's recommendations. The extension times can be found above the respective lane (in minutes). Only Phusion® DNA Polymerase amplified this amplicon in high yields.
Licenses / Patents / Disclaimers:
Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. Phusion® is a registered trademark and property of Thermo Fisher Scientific.
Notice to purchaser: Limited license.
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents owned by BIO-RAD Laboratories, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product.
The purchase price of this product includes a limited, non-transferable license under a patent owned by New England Biolabs, Inc., to use this product. No other license under this patent is conveyed expressly or by implication to the purchaser by the purchase of this product.
Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502). New England Biolabs, Inc. gives the Buyer / User a non-exclusive license to use the aptamer-based Phusion Hot Start Flex DNA Polymerase for RESEARCH PURPOSES ONLY. Commercial use of the aptamer-based Phusion Hot Start Flex DNA Polymerase requires a license from New England Biolabs, Inc. Please contact [email protected] for more information.
For more information, see Technical Resources or neb.com. Information on naming rights can be found here.
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