PCR Related Products.

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Polymerase Chain Reaction

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Presentation transcript:

PCR Related Products

Current Products Thermostable DNA polymerases TaqExtender Pfu PfuTurbo TaqPlus Precision TaqPlus Long Taq TaqExtender Perfect Match Opti-Prime

Components of PCR Thermostable DNA polymerase Reaction Buffer Primers Template DNA dNTPs Water Cycling Conditions Each component affects the quality of the PCR

PCR Qualities Yield Fidelity Specificity Length These are the qualities that we can measure and optimize. Usually one quality has to be sacrificed to improve another.

Thermostable DNA polymerases Pfu PfuTurbo TaqPlus Precision TaqPlus Long Taq

error rate extension Recommended PCR Target (x10 ) time Length (kb) -6 ) time Length (kb) required (min/kb vector genomic target) targets targets PfuTurbo DNA 1.3 + 0.2 1 0-10 0-6 polymerase 2 0-15 0-10 Pfu DNA 1.3 + 0.2 2 0-10 0-4 polymerase TaqPlus 3.9 + 1.3 1 0-15 0-10 Precision system TaqPlus Long 7.6 + 1.2 0.5-1 0-35 0-18.5 system Taq DNA 8.0 + 3.9 1 0-10 0-5 polymerase

Functional Domains of Pfu DNA Polymerase 5’-3’ exo DNA-dependent DNA polymerase 3’-5’ exo 1 126 329 775 non- functional proofreading DNA synthesis activity

Fidelity Assay PCR Mutations are detected in the lacI gene not single round extension Mutations are detected in the lacI gene Color detection from Big Blue system Results have been published in peer reviewed journals

Characteristics of Pfu High Fidelity Native Hot Start activity High Thermostability Slow polymerization rate Available as native and cloned enzyme

Accuracy of Polymerases in PCR

Extension Time Pfu requires 2 min/kb Minutes of extension time/kb of PCR target M 0.25 0.5 1 1.5 2 2.5 3 3.5 4 Taq Pfu

Activity vs Temperature Pfu and Taq percent activity 25 35 50 60 75 95 Incubation Temperature (degrees C)

Pfu: Hot Start Every Cycle M T TS ATG TMg Pfu

Polymerase Thermostability DNA Polymerase Polymerization Opt Half Life 95oC Pfu 70-80oC 19 hours Taq 70-80oC 1 hour

Characteristics of PfuTurbo Improved version of Pfu Same high fidelity as Pfu Higher PCR product yield than Pfu alone Can use 1 min/kb extension times Works well with low amounts of DNA template

PCR Enhancing Factor (PEF) Isolated from Pyrococcus Lacks DNA polymerase and exonuclease activities Information on identity/mechanism of action is proprietary

PfuTurbo vs Other Polymerases M PT P V DV Pw K T M PT P V DV Pw K T M 5.2 kb 0.9 kb

PfuTurbo vs Pfu M T P T P T P T P T P 8 kb 6.1 kb 5.2 kb 1.9 kb 0.9 kb

Characteristics of TaqPlus Precision Blend of Taq and Pfu High Yield High Fidelity Length PCR Buffer optimized for both yield and fidelity

Accuracy of Polymerase Blends in PCR

PCR with TaqPlus Precision vs Taq Taq TaqPlus Precision M 2 5.2 5.4 10 15 M 2 5.2 5.4 10 15 kb

Characteristics of TaqPlus Long Blend of Taq and Pfu Long targets Fidelity similar to Taq Two optimal buffers supplied high salt buffer helps prevent smearing in long target amplification low salt buffer

PCR with Taq vs TaqPlus Long

Qualities of Taq Taq2000 is the recombinant Taq Native Taq is also available Taq is trusted by customers Taq has been well studied

TaqExtender The Pfu component of TaqPlus Long Can be added to any Taq to make a polymerase blend

Perfect Match PCR Enhancer Taq2000 native Taq M - + - +

Opti-Prime PCR Optimization Kit 12 buffers matrix of pH, Mg, KCl Adjuncts DMSO, formamide, BSA, glycerol, ammonium sulfate, Perfect Match Recommended for use with Taq

Related PCR products RoboCycler Nucleotides and primers gradient Nucleotides and primers StrataPrep PCR purification kit Cloning Vectors PCR-Script Kits Affinity protein expression kit QuikChange site-directed mutagenesis kit Cyclist DNA sequencing kits