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Technotes Choose Right Enzyme for PCR TechNotes Choose Right Enzyme for PCR PCR Selection guide, presented is the easy and not time-consuming way to choose enzyme for most of PCR applications, including special PCR: Hi-Fi PCR, “Fast”-PCR or “Direct-Blood” PCR. Taq SmarTaq SmarTth HStorm UltraSmar HF- HS- Taq Taq Fuzz Fuzz Properties 5’->3’ activity + + + + + + + 3’->5’ exo activity - - - - - + + 5’->3’ exo activity + + + - + - - Amplicon Size <5Kb >5Kb >7Kb <5Kb >5Kb >15kb >15kb Resulting Ends A-tail A-tail A-tail A-tail A-tail blunt blunt Units per reaction (50ul) 1,25-2,5 1,25-2,5 1,25-2,5 1-2 1-2,5 0,5-1 0,25-1 Application Reverse Transcription - - + - - weak weak Hot-start PCR - + + + + - + Fast PCR - +/- - + + + + Direct-blood PCR - - - + + + + LD PCR - - - - - + + GC–rich templates - + + + + + + AT-rich templates + + + + + + + High Fidelity PCR - - - - - + + High Yield + + + + + + + Real-Time PCR +/- + + + + + + Taq-Man probe qPCR + + + - + - - Presentation Enzyme in Glycerol Buffer + + + + + + + Enzyme in Low Glycerol Buffer - + - - + + + 2,5X-5X Master Mix - 2,5-5X 2,5-5X 2,5X 2,5X 2,5X 2,5X Ready-to load Master Mix - + - + + + + TAQ DNA Polymerase SmarTth Polymerase Source: Thermus aquaticus YT1 strain Source: Thermus thermophilus HB8 strain, Mouse monoclonal antibodies (two clones) TAQ DNA Polymerase is a thermostable 94 kDa DNA Polymerase purified fromE. coli PVG-AI recombinant SmarTth DNA Polymerase is complex mixture of a strain expressing Thermus aquaticus polymerase thermostable 94 kDa Tth DNA Polymerase purified gene. from E. coli PVG-AI recombinant strain expressing Thermus thermophilus HB8 polymerase gene and The enzyme catalyzes polymerization of nucleotides two clones of specific monoclonal antibodies. into duplex DNA in the 5’-3’ direction in presence of Mg2+ ions. The enzyme possesses also a 5’-3’ SmarTth is inactive under conditions of amplification exonuclease activity. Amplification of target DNA reaction preparation. It provides improved specificity fragments from 100 b.p. to 5000 b.p. can be achieved when compared to standard DNA polymerases. with this enzyme. SmarTth can eliminate amplification artifacts such as primer-dimer formation and mispriming. Applications: - Routine PCR, Primer extension, DNA sequencing SmarTth also has reverse-transcription activity in presence of Mn2+ ions, as regular Tth polymerase. An advantage of SmarTth is the absence of SmarTaq DNA Polymerase additional heating step for polymerase activation. Heat activation of enzyme occurs during the first Source: Thermus aquaticus YT1 strain, Mouse denaturation step. An active complex of SmarTth o monoclonal antibodies dissociates automatically over +70 C, allowing activating DNA polymerase. SmarTaq DNA Polymerase is complex mixture of a thermostable 94 kDa Taq DNA Polymerase purified from E. coli PVG-AI recombinant strain expressing Thermus aquaticus polymerase gene and highly UlrtaSmarTaqPolymerase specific monoclonal antibodies. Source: E. coli strain expressing the cloned SmarTaq is inactive under conditions of amplification UlrtaSmarTaqDNA Polymerase gene. Specific reaction preparation. It provides improved Monoclonal antibodies. specificity and high yield amplification, even with low copy number and complex DNA templates, UlrtaSmarTaq DNA Polymerase is complex mixture when compared to standard DNA polymerases. of a thermostable 94 kDa modified fused Taq SmarTaq can eliminate amplification artifacts, DNA Polymerase special constriction purified such as primer-dimer formation and miss-priming. from E. coli strain expressing Thermus aquaticus polymerase gene and highly specific monoclonal An advantage of SmarTaq is the absence of antibodies. additional heating step for polymerase activation. Heat activation of enzyme occurs during the first UlrtaSmarTaq as hot-start enzyme inactive under denaturation step. An active complex of SmarTaq conditions of amplification reaction preparation dissociates automatically over +70 oC, allowing (room temperature). It provides improved activating DNA polymerase, preventing miss-priming specificity and high yield amplification, even with during amplification. low copy number and complex DNA templates, when compared to standard DNA polymerases. UlrtaSmarTaq can eliminate amplification artifacts, such as primer-dimer formation and misspriming. 2 TechNotes | Choose Right Enzyme for PCR HF-Fuzz Polymerase An advantage of UlrtaSmarTaqis the absence of Source: E. coli strain expressing the cloned additional heating step for polymerase activation. HF-Fuzz DNA Polymerase gene. Heat activation of enzyme occurs during the first denaturation step. An active complex of HF-Fuzz DNA Polymerase is a unique artificial UlrtaSmarTaqdissociates automatically over +70 oC, enzyme created on the basis of intellectual protein allowing activating DNA polymerase, preventing design planning by genetic engineering technique. miss-priming during amplification. The enzyme possess high fidelity feature. The processivity of the enzyme is very high, so the In the contrast to “regular” Taq-based enzymes, which combination of processivity with fidelity results are inhibited by blood components modifications in dramatically increased yield of PCR products, entered into UlrtaSmarTaq polymerase gene very high sensitivity of PCR tests, ability to amplify allowed to amplify DNA sequence directly from “difficult” templates.This dramatic increase in whole blood, stabilized with the commonly used procesivity results not only in shorter extension agents (heparin, citrate or EDTA). times, but in more robust amplification and the ability to amplify long templates really fast. UlrtaSmarTaq DNA Polymerase possesses the 5’->3’ DNA polymerase activity, 5’->3’ exonuclease HF-Fuzz DNA Polymerase possesses the 5’->3’ activity and temperature-depended strand- DNA polymerase activity, 3’->5’ exonuclease activity displacement activity and generates A-tailed ends in and temperature-depended strand-displacement the amplification products. activity and generates blunt ends in the amplification products. UltraSmarTaq could be used in “fast-PCR” mode dramatically reducing time of PCR run – less than HF-Fuzz provides: 40 min instead of 1,5-2 hours for the “regular” PCR. • Superior fidelity - 50x improvement compared to UlrtaSmarTaq could be used in all “real-time” PCR Taq polymerase; applications including probe-based PCR (Taq-Man • Excellent performance across a wide range of probes, beacons, etc.) or with intercalating dyes “difficult” templates; (SYBR Green I / Eva Green) • Long range amplification of complex targets - > 10 kb from genomic DNA; UlrtaSmarTaq provides: • High speed - reduce reaction times. • Excellent performance across a wide range of • dUTP poisoning resistance “difficult” templates; • Resistance to blood containing DNA samples • Long range amplification of complex targets (up to 20% of blood) - > 5 kb from genomic DNA; • High speed - reduce reaction times. • dUTP poisoning resistance • blood components poisoning resistance • “fast-PCR” mode amplification • excellent reproducibility in all types of “real- time” PCR TechNotes | Choose Right Enzyme for PCR 3 TechNotes | Choose Right Enzyme for PCR HS-Fuzz Polymerase Source: E. coli strain expressing the cloned very high sensitivity of PCR tests, ability to amplify HS-Fuzz DNA Polymerase gene. Anti-Pfu mouse “difficult” templates. This dramatic increase in monoclonal antibodies processivity results not only in shorter extension times, but in more robust amplification and the HS-Fuzz DNA Polymerase is a unique artificial ability to amplify long templates really fast. enzyme created on the basis of intellectual protein design planning by genetic engineering technique. HS-Fuzz DNA Polymerase possesses the 5’->3’ DNA polymerase activity, 3’->5’ exonuclease activity HS-Fuzz enzymatic activity at ambient temperature and temperature-depended strand-displacement is blocked by highly specific monoclonal antibodies activity and generates blunt ends in the amplification against HS-Fuzz polymerase. Antibodies allows to products. switch enzymatic activity during first denaturation step of PCR run, but keeps HS-Fuzz inactive during HS-Fuzz provides: PCR reaction setup. • Superior fidelity - 50x improvement compared to Taq polymerase; This feature results in improvement of specificity • Excellent performance across a wide range of of PCR and increasing of yield of PCR product, “difficult” templates; preventing miss priming and PCR artifacts formation • Long range amplification of complex targets – primer-dimers. - > 10 kb from genomic DNA; • High speed - reduce reaction times. The enzyme possess high fidelity feature. The • dUTP poisoning resistance processivity of the enzyme is very high, so the • Resistance to blood containing DNA samples combination of processivity with fidelity, results (up to 20% of blood) in dramatically increased yield of PCR products, Intelligate 6th floor, Joukahaisenkatu 6 FI-20520 Turku, FINLAND Tel. +358 2 512 0900. Fax +358 2 512 0909 E-mail: [email protected] Internet: http://www.hytest.fi NovemberHyTest 2013.
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