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Upgrade to the gold standardfor high-fidelity PCRNEWSince their introduction in 2003,Thermo Scientific Phusion High-FidelityDNA Polymerases have established anew standard for high-fidelity PCR. (1,2)Phusion High-Fidelity DNA Polymerase contains a DNA-binding domain fused toa Pyrococcus-like proofreading polymerase. Due to this unique fusion technique,Phusion DNA Polymerases generate PCR products with accuracy and speedunattainable with a single enzyme, even with difficult templates. In addition,Phusion DNA Polymerases are tolerant of various inhibitors allowing for robustamplification of PCR products with minimal optimization. For hot start PCR,Thermo Scientific TM Phusion TM Hot Start II High-Fidelity DNA Polymerase is anideal choice allowing extreme specificity and improved robustness.The processivity* of Phusion DNA Polymerases is approximately 10-fold greaterthan that of Pfu DNA polymerase and twice that of Taq DNA polymerase. Thisextremely high processivity results in shorter extension times, more robustamplification and the ability to amplify long templates (up to 20 kb) in a fractionof time. Phusion DNA Polymerases also produce higher yields with lower enzymeamounts than traditional proofreading polymerases.The new Thermo Scientific TM Phusion TM Green format is acombination of Phusion DNA Polymerase and 5x Green ReactionBuffer. The buffer includes a density reagent and two trackingdyes for direct loading of PCR products on gels. The greenbuffer does not interfere with the performance of Phusion DNAPolymerase and is compatible with downstream applicationsincluding DNA sequencing, ligation and restriction digestion.Features• Accuracy – the highestfidelity thermostablepolymerase (52x Taq)• Robustness – fewerreaction failures and minimaloptimization• Speed – increasedprocessivity allows shorterreaction times (extension15-30 s/kb)• High yields – increasedproduct yields withminimal enzyme amounts(0.5‐1 U/50 µL reaction)• Specificity – unique hot starttechnology with zero-timereactivation reduces nonspecificamplification andprimer degradation• Direct loading of PCRproduct on gels – PhusionGreen and Phusion GreenHot Start II DNA Polymeraseshave loading dyes includedinto reaction buffersApplications• High-fidelity PCR• Fast PCR• Hot start PCR• Long range PCR(up to 20 kb)• High-throughput PCR1. D.G. Gibson et al., (2008) Complete chemical synthesis,assembly, and cloning of a Mycoplasma genitalium genome.Science 319, 1215-1220.2. D.G. Gibson et al., (2010) Creation of a bacterial cell controlledby a chemically synthesized genome. Science 329, 52-56.* Processivity measures the number of nucleotides the enzymecan incorporate into a growing DNA strand at one bindingevent during the extension step.

Extreme fidelityIn many molecular biology applications including cloning, site-directedmutagenesis and DNA translation, it is crucial to preserve the accurateDNA sequence during PCR amplification. An incorrectly incorporatednucleotide may change the respective codon and result in the addition ofthe wrong amino acid during translation. This, in turn, can affect foldingand functional properties of the protein. Alternatively, deletion of a singlenucleotide can destroy the correct reading frame.Phusion DNA Polymerases have the highest fidelity of any availablethermostable polymerase. The error rate of Phusion DNA Polymerase asdetermined by a modified lacI-based method 3 is approximately 50-foldlower than that of Taq DNA polymerase and six-fold lower than that of PfuDNA polymerase (see graph below).The low error rate of Phusion DNA Polymerase was recently confirmedin studies using 454 sequencing 4 and Illumina sequencing methods 5(Table 1).Successful amplification of longtargetsPhusion DNA Polymerases are the ideal choice for amplification of longtemplates. Extremely high enzyme processivity allows amplification of thewidest variety of template sizes. Amplicons up to 20 kb are produced withhigh yields, short cycling times and higher fidelity.20 kb (viral) 7.5 kb (genomic)M PG P A B C PG P A B C MPG: Phusion Green High-Fidelity DNA PolymeraseP: Phusion High-Fidelity DNA PolymeraseA-C: Proofreading DNA polymerases from other suppliers52x Taq32x TaqSuperior yields of long PCR productsA 20 kb fragment from λ DNA and 7.5 kb fragment from humangenomic DNA was amplified with Phusion DNA Polymerases andproofreading DNA polymerases from other suppliers.Phusion DNAPolymerasesPfu-basedfusion DNApolymerase8x Taq7x TaqPfu KOD TaqRelative fidelity values of different DNApolymerases. Fidelity = 1 / error rate.KOD(%)PhusionHF (%)Pt Taq(%)ExpandHF (%)1x TaqFastStartHF (%)Sequal PrepLong (%)Pfu UltraHF (%)Robust amplificationwith less enzymeDue to the unique structure of the enzyme, Phusion DNA Polymerasesare highly efficient. When compared to conventional polymerases,significantly fewer units of the enzyme are required for any PCRamplification. Speed and efficiency result in high product yields inminimal time. In addition, Phusion polymerases are highly robustminimizing the need for reaction optimization.Overall errorrate a 0.21 0.11 0.34 0.25 0.23 0.29 0.23Insertions 0.10 0.07 0.14 0.11 0.11 0.11 0.12Pfu(5 U)modified Pfu(2.5 U)Phusion DNAPolymerase (1 U)Deletions 0.06 0.02 0.08 0.07 0.05 0.06 0.05Substitutions 0.01 0.01 0.07 0.04 0.03 0.07 0.011 min1 min 30 s3 min 50 s7 min 40 s1 min1 min 30 s3 min 50 s7 min 40 s1 min1 min 30 s3 min 50 s7 min 40 sDots or Dot b 0.04 0.01 0.05 0.04 0.04 0.05 0.05Table 1. Error rates determined by 454 sequencing for seven differentDNA polymerases following PCR amplification of four different exonsfrom the human TP53 oncogene. The clonal TP53 plasmid was used as astarting template.a Error rate, number of errors (miscalled bases, inserted or deleted bases) divided by total number of bases.b Dots or Dot, three successive negative flows during 454 sequencing.Table reprinted with the permission from the corresponding author and the journal editor.3. B.F. Frey & B. Suppmann (1995) Demonstration of the Expand PCR System’s greater fidelity and higher yields witha lacI-based fidelity assay. Biochemica 2, 34-35.4. Vandenbroucke et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing:experiences and considerations for successful applications. Biotechniques. 53:167-177.5. Kinde et al. (2011) Detection and quantification of rare mutations with massively parallel sequencing. PNAS.108(23):9530–9535.Less enzyme – superior yieldA 3.8 kb fragment from human beta globin gene was amplified with threedifferent DNA polymerases. Phusion DNA Polymerase was able to amplifythe 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 producedhigher yields than 2.5 or 5 units of the Pfu DNA polymerases.

Extreme specificity hot start PCRPhusion Hot Start II High‐Fidelity DNA Polymerase is the most accuratehot start DNA polymerase on the market. It combines the PhusionDNA Polymerase and a reversibly bound, specific Affibody TM ligand.The Affibody ligand inhibits the activity of the DNA polymerase atroom temperature and thus prevents the amplification of nonspecificproducts. The reaction set-up can be done at room temperatureenabling its use in high throughput robotics.The Affibody ligand also inhibits the 3´5´ exonuclease activity ofthe polymerase, preventing degradation of primers and template DNAduring reaction set-up. At polymerization temperatures, the ligand isreleased, rendering the polymerase fully active. Phusion Hot Start IIDNA Polymerase does not require a separate activation step in the PCRprotocol as it is immediately reactivated at high temperatures.1.7 kb 2.2 kb 2.3 kb (GC-rich)M P A B C D M P A B C D M P A B C D MFast PCR with high fidelityPhusion DNA Polymerases incorporate more nucleotides per binding eventas compared to other polymerases. This high processivity allows extremelyshort extension times and consequently reduced protocol times. Shortestprotocol times can be achieved with Thermo Scientific TM Phusion TM FlashHigh-Fidelity PCR Master Mix, a product developed specifically for fast PCR.605040302010min0Phusion FlashFast Taq polymeraseFast cyclerPfu-based fusion polymerasePhusion FlashFast Taq polymerasePfu-based fusion polymeraseConventional cyclerShorter PCR run times with Thermo Scientific PhusionFlash Master MixP: Phusion Hot Start II High-Fidelity DNA PolymeraseA-D: Proofreading hot start DNA polymerases from major suppliersThermo Scientific Phusion Hot Start II DNA Polymerase providesextreme specificity and abundant yieldsFive proofreading hot start DNA polymerases from major suppliers wereused to amplify 1.7-2.3 kb fragments from human genomic DNA. PhusionHot Start II DNA Polymerase provided high yields of specific productswhereas all other enzymes delivered zero or low yields, with some alsoamplifying non-specific products.Phusion Flash60 s45 s30 s20 s10 sPfu-based fusionpolymerase60 s45 s30 s20 s10 sFast Taqpolymerase60 s45 s30 s20 s10 sRobust amplification even withhigh GC content templatesOptimized buffer system in synergy with the high enzyme processivityenables Phusion DNA Polymerase to amplify a broad range of DNAtemplates with different sequence contexts. Efficient high-fidelityamplification has been demonstrated with difficult-to-amplify targetsincluding those with 85% GC content.Extreme speed and high yields with Thermo ScientificPhusion Flash Master MixA 1.5 kb human cathepsin K gene was amplified with threedifferent polymerases using varying extension times (10‐60 s)with Thermo Scientific Piko Thermal Cycler. Only PhusionFlash Master Mix was able to amplify the 1.5 kb gene withextremely short extension times of 10 and 20 s. It alsoproduced superior yields of specific product compared toother enzymes tested.M P A P A P A P A M38% 65% 71% 85%P: Phusion Green High-Fidelity DNA PolymeraseA: Competing high-fidelity DNA polymerasefrom other supplierRobust amplification of DNAfragmentsFour DNA fragments of different GCcontent were amplified. PhusionGreen DNA Polymerase producedall four amplicons with highyields. In contrast, a competinghigh‐fidelity DNA polymerase wasnot able to efficiently produceGC‐rich product.

Thermo Scientific Phire Green reaction buffer – superior enzymeperformance and ultimate convenience477 bp 1.1 kb 1.7 kb 7.5 kbM P A B P A B P A B P A B MP: Phire Green Hot Start II DNA PolymeraseA-B: Hot start Taq polymerases from other suppliersA 1x reaction mixture containingThermo Scientific Phire GreenReaction Buffer.A – unseparated in a wellB – blue and yellow dyesfollowing electrophoresisB Thermo Scientific Phire Green Hot Start IIDNA Polymerase amplifies longer fragments thanany hot start TaqFive human genomic DNA fragments of different lengthswere amplified with three different hot start DNApolymerases. Phire Green Hot Start II DNA Polymeraseproduced all five amplicons with high yields. Thecompeting hot start Taq DNA polymerases producedsignificantly lower yields and failed to amplify the7.5 kb fragment.Enzyme characteristics and formatsPhusion High-FidelityDNA PolymerasePhusion Hot Start II High-Fidelity DNA PolymerasePhusion Flash High-FidelityDNA PolymerasePhire Hot Start IIDNA PolymeraseBlunt or 3’A end Blunt Blunt Blunt BluntCharacteristicsTarget length ≤ 20 kb ≤ 20 kb ≤ 20 kb ≤ 7.5 kbHot start No Yes Yes YesRecommended extension time 15-30 s/kb 15-30 s/kb 15 s/kb 10-15 s/kbFidelity vs. Taq 52x 52x 25x 2xAvailable asEnzyme 1 – Green Buffer 2 – Master mix 3 – –Complete kit 4 – – –1. Enzyme: DNA polymerase, buffer(s), DMSO and MgCl 22. Green Buffer: DNA polymerase supplied with a Green buffer that includes density reagent and two tracking dyes for direct PCR product loading on gel.3. Master mix: 2x master mix4. Complete kit: All the necessary PCR reaction components including control template and primers.

The complete solution –high-performance PCRThermo Scientific High-Performance system combines best-in-class DNApolymerases, PCR instruments and reaction vessels into a truly integratedsolution for PCR. This powerful combination enables savings in time,space and cost, allowing DNA amplification with the highest accuracy andsignificantly shorter protocol times when compared to any other system.Phusion High-FidelityDNA Polymerases orPhire Hot Start II DNAPolymerasesPiko Thermal CyclerUltra Thin Wall (UTW)reaction vesselsSpeedSignificantly faster than any other combination.FidelityPhusion DNA Polymerases offer superior accuracy over Taq- andPfu-based systems.YieldsEfficient template amplification results in high yields of PCR products.SpecificityReduced levels of primer-dimers and false-primed products.Direct loadingNEW Phusion and Phusion Green DNA Polymerases contain loadingdye for direct loading of PCR products on gels!Everything foryour ThermoScientific PCRworkflowSamplepreparation• Nucleic AcidPurification Kits• Plasticconsumables• Storage platescDNAsynthesiscDNAsynthesis• Thermo ScientificMaxima ReverseTranscriptases• Thermo ScientificMaxima First StrandcDNA Synthesis Kits• ThermoScientificRiboLockRNAse InhibitorAmplification• Phusion High FidelityDNA Polymerases• Phire Hot Start IIDNA Polymerases• Piko ThermalCyclers• Plasticconsumablesthermoscientific.com/pcr

Order detailsProduct Description Quantity Cat.No.Phusion High-Fidelity DNA Polymerases,Master Mixes and KitsPhusion High-Fidelity DNA Polymerase100 U F-530S500 U F-530LPhusion Green High-Fidelity DNA100 U F-534SPolymerase500 U F-534LPhusion High-Fidelity PCR Master Mixwith HF BufferPhusion High-Fidelity PCR Master Mixwith GC BufferPhusion Flash High-Fidelity PCR MasterMixPhusion High-Fidelity PCR KitPhusion Hot-Start II High-Fidelity DNAPolymerasePhusion Green Hot Start II High-FidelityDNA PolymerasePhusion RT-PCR KitPhusion Site-Directed Mutagenesis KitPhire Hot Start II DNA PolymerasePhire Hot Start II DNA PolymerasePhire Green Hot Start II DNA Polymerase100 × 50 µL rxns F-531S500 × 50 µL rxns F-531L100 × 50 µL rxns F-532S500 × 50 µL rxns F-532L100 × 20 µL rxns F-548S500 × 20 µL rxns F-548L125 × 20 µL rxns F-553S500 × 20 µL rxns F-553L100 U F-549S500 U F-549L100 U F-537S500 U F-537L20 rxns F-546S100 rxns F-546L20 rxns including10 control rxnsF-541200 × 50 µL rxns(or 500 × 20 µL rxns)1000 × 50 µL rxns(or 2500 × 20 µL rxns)200 × 50 µL rxns(or 500 × 20 µL rxns)1000 × 50 µL rxns(or 2500 × 20 µL rxns)F-122SF-122LF-124SF-124LDirect PCR KitsPhire Plant Direct PCR Kit 200 × 50 µL rxns F-130Phire Animal Tissue Direct PCR Kit 200 × 50 µL rxns F-140Phusion Human Specimen Direct PCR Kit 200 × 20 µL rxns F-150Phusion Blood Direct PCR Kit100 × 20 µL rxns F-547S500 × 20 µL rxns F-547LIn addition to Phusion and PhirePCR enzymes and kits, the ThermoScientific PCR portfolio includesPCR instruments, reaction vesselsand alternative DNA polymerasesfor various applications.• Learn more atthermoscientific.com/PCR“After realizing that we could get thesame number of cycles in roughly aquarter of the time (and at only slightlyhigher per unit cost), we changedexclusively to Phusion [Polymerase].Matt W. Ford, PhD student,Department of Biological Sciences,Idaho State University, USA.”© 2013 Thermo Fisher Scientific Inc. All rights reserved. Affibody is a registered trademark of Affibody AB, Sweden. All other trademarks are the property ofThermo Fisher Scientific Inc. and its subsidiaries. Legal/license information is provided in specific product insert.EuropeUnited StatesCanadaCustomer Servicecs.molbio.eu@thermofisher.comCustomer Servicecs.molbio@thermofisher.comCustomer Servicecs.molbio@thermofisher.comTechnical Supportts.molbio.eu@thermofisher.comTechnical Supportts.molbio@thermofisher.comTechnical Supportts.molbio@thermofisher.comMB1318Tel 00800 222 00 888Fax 00800 222 00 889Tel 800 235 9880Fax 800 292 6088Tel 800 340 9026Fax 800 472 8322