Highfidelity dna polymerases are important for applications in which the dna sequence needs to be correct after amplification. In addition to being faster than taq, phusion also makes fewer errorskb on average at least, compared to a standard taq polymerase there are various high fidelity variants out there. Using phusion hot start ii highfidelity dna polymerase, amplification proceeds without the production of nonspecific products due to the combination. It is frequently used in the polymerase chain reaction pcr, a method for greatly amplifying the quantity of short segments of dna. Overlap extension pcr, phusion polymerase or taq polymerase. A testing various dna polymerases for the amplification of a 1 kb fragment with 70% gccontent. A variant of this protein with an additional 65 amino acid changes is sold as phusion polymerase by new england biolabs. This is where dna polymerase enters the world of forensics. Source organism phusion is extracted from extremophilic archaea. The polymerase is tolerant against various inhibitors allowing stable amplification with minimized assay optimization. Finnzymes phusion highfidelity dna polymerase offers extreme performance for all pcr applications. Polymerases for isothermal amplification new england biolabs. Phusion hot start dna polymerase guidelines for pcr. Each polymerase has different features, resulting from origin and genetic modification.
Feb 01, 2015 as highfidelity dna polymerases, both phusion and kod dna polymerase have the ability to synthesize modified nucleotides into dna duplexes. Phusion highfidelity dna polymerase, new england biolabs. Using a lacibased method modified from previous studies1, the error rate of phusion dna polymerase in. The following are supplementary data to this article. Difference between phusion and taq polymerase compare. Thermo scientific phusion u dna polymerase is a novel engineered high fidelity enzyme developed using fusion technology. How to set up a pcr reaction with phusion dna polymerases. For the researcher, that translates to easeofuse and convenience. Control template and primers are provided for 20 control reactions. Therefore, an 11 to 12 basepair length of rna an rna primer is made at the beginning of each new strand of dna. A novel assay for dnadependent dna polymerase activity.
Phusion is a dna polymerase isolated from pyrococcus furiosus and is mainly used in cloning experiments to increase the fidelity taq dna polymerase is the standard dna polymerase used in the polymerase chain reaction pcr, and it is isolated from the thermostable bacterium, thermus aquaticus. L template pet24a vector with the kod dna pol gene, 500 nm forward primer 5dtttgcactgggtcgtgatg, 500 nm reverse primer 5dcagttccagtgcacccggc, 200. Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications. New phusion green format is a combination of phusion dna polymerase and 5x phusion green buffers.
Phusion dna polymerase is an ideal choice for cloning and can be used for long or difficult gcrich amplicons. Phusion dna polymerase brings together a novel pyrococcuslike enzyme with a processivityenhancing domain and generates pcr products with accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates. Overview of thermostable dna polymerases for classical pcr. Thermocycling conditions were standard as recommended in the protocol except for extension time. Dna polymerase cannot initiate new strands of nucleic acid synthesis because it can only add a nucleotide onto a preexisting 3.
However, the rational choice of the adequate polymerase depends on the application itself. In the structure of the dna polymerase from rb69 five domains are arranged around a central hole. Difference between phusion and taq polymerase compare the. Learn more about phusion highfidelity dna polymerase. A highthroughput assay for quantitative measurement of.
Phusion dna polymerase may be diluted in 1x hf or gc buffer just prior to use in order to reduce pipetting errors. Phusion dna polymerases offer robust performance with short protocol times, even in the presence of pcr inhibitors, and generate higher yields with lower enzyme amounts than other dna polymerase. During the genomics era, the use of thermostable dna polymerases increased greatly. Amplification of a 500bp fragment using pfuultra highfidelity dna polymerase results in errors in less than 0. Here we describe the generation of thermostable mutants of the large fragment of thermus aquaticus dna polymerase klentaq. It generates blunt ends in the amplification products. Systems biology in toxicology and environmental health, 2015. The tms should be calculated with the nearestneighbor method because results from primer tm calculations can vary significantly depending on the method. Additionally, phusion dna polymerase is capable of amplifying long templates.
Dna replication is semiconservative arthur kornberg discovered dna dependent dna polymerase used an in vitro system. Taq dna polymerase is relatively processive 60% that of phusion, but the overall fidelity of the mixture is only 3. Phusion dna polymerase possesses 53 polymerase activity, 35 exonuclease activity and will generate bluntended products. Phusion dna polymerase, 5x phusion hf buffer, 5x phusion gc buffer, dntp mix, mgcl2 solution, control lambda dna, 1. Phusion highfidelity dna polymerase new england biolabs. At the top of the page youll find a map of all positions of known mutants. Pmc images search for phusion highfidelity dna polymerase figure 1 engineered proofreading dna polymerases have good performance. Roche, basel, switzerland, which is mostly taq dna. The purchase price of this product includes a limited, nontransferable license under u. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. Dna polymerase is a ubiquitous enzyme that synthesizes. We present data on the enhanced thermostability of staq dna polymerase over taq dna.
The effects of seven major types of metal oxide nps fe2o3, zno, ceo2, fe3o4, al2o3, cuo, and tio2 on pcr replication via a lowfidelity dna polymerase ex taq and a highfidelity dna polymerase phusion were tested. The phusion highfidelity dna polymerase should be pipetted carefully and gently as the high glycerol content 50% in the storage buffer may otherwise lead to pipetting errors. By fusing a particular doublestranded dna binding domain to the polymerase, its processivity can be increased 10fold. Lynch, in physiology of the gastrointestinal tract sixth edition, 2018. Phusion highfidelity dna polymerase phusion hot start ii highfidelity dna polymerase phusion flash highfidelity dna polymerase phusion u hot start dna polymerase phusion u multiplex pcr master mix characteristics blunt or 3a end blunt blunt blunt blunt blunt target length, genomicphage dna. Dec 05, 2014 in this video we will show you how to set up a pcr reaction usingthermo scientific phusion highfidelity dna polymerase.
Search for dna polymerase activity using an assay incorporate radioactive building blocks. New insights into the quikchangetm process guide the use of. Phusion dna polymerases thermo fisher scientific us. Phusion highfidelity dna polymerases have become the choice for even demanding applications like massively parallel highthroughput sequencing of whole genomes. The polymerase is based on pfu with a fused dna binding domain. Phusion highfidelity dna polymerase high performance for all. Guaninerich sequences inhibit proofreading dna polymerases. Thermo scientific phusion sitedirected mutagenesis kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid dna. New insights into the quikchangetm process guide the use. However, this technique is often plagued with its low specificity and sensitivity. Pdf production and evaluation of taq dna polymerase. This page presents all the information in polbase for phusion.
Pcr using phusion high fidelity dna polymerase biocompare. The phusion highfidelity pcr kit contains a sufficient supply of phusion highfidelity dna polymerase, phusion hf and gc buffers, deoxynucleotides, magnesium chloride, dmso, and dna size standard to perform 50 reactions small or 200 reactions large. The accuracy with which dna polymerase can replicate a template dna sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. Ultra dna polymerase, taq polymerase jena bioscience. Variants of a thermus aquaticus dna polymerase with increased. Both phusion hf buffer and phusion gc buffer provide 1. In this enzyme the fingers domain consists largely of two very long. Thermo scientific phusion green highfidelity dna polymerase. The enzymes polymerase activity was determined by amplifying a 1044 bp long part of the kod dna pol gene using 1x phusion hf buffer new england biolabs, 0. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Mar 30, 2016 250 ng template dna was used in a 50 microliter reaction volume with 1x phusion hf buffer, 500 nm each of forward and reverse primer, 0.
A small sample of dna is multiplied using pcr the polymerase chain reaction, creating a large sample that may be easily analyzed. Comparing with unmodified dntps, the modifications of nucleotides help to prolong the halflife of oligonucleotides introduced into cells or improve the affinity toward targets 36. Pcr reagents are kits that include researchersupplied components, such as purified nucleic acid templates and oligonucleotide primers, along with components available through vwr, such as deoxyribonucleotides dntps, and dna polymerase. The aim of this study was to amplify the gene of this enzyme from a. This is the pcr protocol for phusion highfidelity dna polymerase m0530. Phusion highfidelity dna polymerases product listing product overview manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications. Jan 30, 2015 the linear dna molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in e. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and phusion dna polymerase for sitedirected mutagenesis. Grich inhibitory sequencecontaining primers can be used in pcr at a lower concentration to amplify its target dna fragment. Its unique structure, a novel pyrococcus like enzyme fused with a processivity enhancing domain, increases fidelity and.
Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Phusion hot start ii green master mix a and proofreading hotstart 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. The processivity of phusion dna polymerases is approximately 10fold greater than that of pfu dna polymerase and twice that of taq dna polymerase. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear dna molecules with homologous ends. All that is required is the addition of template, primers and water. Phusion highfidelity dna polymerase for high speed and high fidelity pcr, 52x greater fidelity than taq. This pfu variant has the sso7d processivityenhancing domain attached that increases its speed and processivity. This protocol is for expressing and purifying the pfusso7d polymerase from e.
Since the leading strand is synthesized as a single piece, there is only one rna primer at the origin. Ultra dna polymerase is a genetically optimized dna polymerase for robust, fast and accurate amplification, even with difficult or gcrich dna templates. The polymerase you use has a significant impact on the efficacy of your pcr, specifically on the product yield, the purity of the product and the faithfulness with which the starting product is transcribed. Phusion hot start dna polymerase has the ability to stabilize primertemplate hybridization. What are the advantagesdisadvantages of phusion vs taq. Thermo scientific phusion highfidelity pcr kit life. Phusion highfidelity dna polymerase is supplied with 5x phusion hf buffer, 5x phusion gc buffer, dmso and 50 mm mgcl2 solution.
The tiny sample is placed in a test tube, and dna polymerase is added to make a copy. Taq polymerase is a thermostable dna polymerase i named after the thermophilic eubacterial microorganism thermus aquaticus, from which it was originally isolated by chien et al. If available, a representative structure is displayed at the. Pdf ttnmgcctc inhibits archaeal family b dna polymerases. Many were identified and describedmainly of the genera thermus, thermococcus and pyrococcus. Phusion highfidelity dna polymerase high performance for.
Sep 20, 2017 phusion dna polymerase is a very high fidelity polymerase which is artificially constructed by dna binding domain to proofreading polymerase from pyrococcus furiosus. Jun 18, 2015 while most may think standard taq is the backbone of pcr, there are many other dna polymerase options out there. Q5 highfidelity dna polymerase new england biolabs. Phusion highfidelity dna polymerase from new england. Dna polymerase activity and strong strand displacement activity but lacks 5. Text links to mutants and digestion products are listed in the mutants section.
Onetaq dna polymerase is an optimized blend of taq and deep vent dna polymerases for use with routine and difficult pcr experiments. Phusion dna polymerase is supplied with standard 5x phusion hf buffer, as well as 5x phusion gc buffer, which can be used for complex or gcrich templates. We enable science by offering product choice, services, process excellence and our people make it. Thermo scientific phusion highfidelity dna polymerase. The speciality of phusion is that it is fused to a dna binding proteins domain thats why its name is phusion, so that it stays attached to the template dna over a longer distance than usual. Monarch nucleic acid purification kits are optimized for maximum performance and minimal environmental impact. Zuo and rabie developed a similar method with taq dna polymerase alone, and reported similarly modest cloning efficiencies. A dna capture probe immobilizes the product that carries an electrochemiluminescent reporter. A novel assay for dna dependent dna polymerase activity conclusion a dna dependent dna polymerase assay has been developed on a novel platform that utilizes disposable carbon electrodes arrayed in microtiter plates. A 2x supermix is now available containing either hf buffer or gc buffer, dntps, and phusion polymerase. The successfully amplified pcr products were subsequently sequenced using highthroughput amplicon sequencing. Most abundant polymerase accounting for 95% of polymerase activity in e. With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes.
Ttnmgcctc inhibits archaeal family b dna polymerases. Anneal at 3c above the lowest tm if the primers are longer than 20nt, else at the tm. Due to a proprietary mutation in the so called dutp binding pocket of phusion, phusion u overcomes an important limitation of proofreading enzymes it is able to incorporate dutp and read through uracil present in dna templates. Thermo scientific phusion highfidelity dna polymerases set a gold standard for high performance pcr. Phusion hot start flex dna polymerase is supplied with standard 5x phusion hf buffer, as well as 5x phusion gc buffer, which can be used for complex or gcrich templates. The phusion highfidelity pcr kit contains a sufficient supply of phusion highfidelity dna polymerase, phusion hf and gc buffers, deoxynucleotides, magnesium chloride, dmso, and dna size standard to perform 50. Phusion highfidelity pcr master mix with hf buffer is a 2x master mix consisting of phusion dna polymerase, deoxynucleotides and reaction buffer that has been optimized and includes mgcl 2. Dna polymerase is the primary enzyme which catalyzes the linking of the 3. Dna polymerases in prokaryotes dna polymerase i this is a repair polymerase and is involved in excision repair with 35 and 53 exonuclease activity and processing of okazaki fragments generated during lagging strand synthesis. Novel properties of recombinant sso7dtaq dna polymerase. The pcr efficiency rate of staq is higher than taq polymerase and can be used to detect dna. Phusion hot start ii dna polymerase does not require any separate activation step in the pcr protocol. Of course, there is very little dna in a dried drop of blood. More than 98% of the colonies transformed with dna produced by phusion dna.
The selectivity of dna polymerases is crucial for many applications. Hence, the pcr mixtures containing either a highfidelity dna polymerase or a conventional dna polymerase were first preincubated for 1 h at a low temperature 35c56c prior to pcr, as it often took 0. Dna polymerase and 18fold lower than taq dna polymerase, making it the highest fidelity enzyme available see table i. Featuring an error rate 50fold lower than that of taq. Polymerase chain reaction pcr has become one of the powerful techniques in biological and medical sciences since 1980s.
Dna polymerases, which are multisubunit enzymes including pol. Impact of metal oxide nanoparticles on in vitro dna. Dna amplification using taq dna polymerase is one of the most widely used techniques in molecular biology and biotechnology. Enzymatic synthesis of modified oligonucleotides by pear. In addition, phusion dna polymerases also produce higher yields with lower enzyme amounts than other dna polymerases.