PCR Related Products. Current Products n Thermostable DNA polymerases –Pfu –PfuTurbo –TaqPlus...

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PCR Related ProductsPCR Related Products

Current ProductsCurrent Products Thermostable DNA polymerasesThermostable DNA polymerases

– PfuPfu– PfuTurboPfuTurbo– TaqPlus PrecisionTaqPlus Precision– TaqPlus LongTaqPlus Long– TaqTaq

TaqTaqExtenderExtender Perfect MatchPerfect Match Opti-PrimeOpti-Prime

Components of PCRComponents of PCR

Thermostable DNA polymeraseThermostable DNA polymerase Reaction BufferReaction Buffer PrimersPrimers Template DNATemplate DNA dNTPsdNTPs WaterWater

– Cycling ConditionsCycling Conditions

PCR QualitiesPCR Qualities

YieldYield FidelityFidelity SpecificitySpecificity LengthLength

Thermostable DNA Thermostable DNA polymerasespolymerases

PfuPfu PfuTurboPfuTurbo TaqPlus PrecisionTaqPlus Precision TaqPlus LongTaqPlus Long TaqTaq

error rate(x10 -6)

extensiontime

required(min/kbtarget)

RecommendedLength

vectortargets

PCR Target(kb)

genomictargets

PfuTurbo DNApolymerase

1.3 + 0.2 1

Pfu DNApolymerase

1.3 + 0.2 2 0-10 0-4

TaqPlusPrecisionsystem

3.9 + 1.3 1 0-15 0-10

TaqPlus Longsystem

7.6 + 1.2 0.5-1 0-35 0-18.5

Taq DNApolymerase

8.0 + 3.9 1 0-10 0-5

Functional Domains of Functional Domains of PfuPfu DNA DNA PolymerasePolymerase

5’-3’exo

3’-5’ exoDNA-dependent DNA polymerase

non-functional

proofreading DNA synthesisactivity

1 126 329 775

Fidelity AssayFidelity Assay

PCR PCR – not single round extensionnot single round extension

Mutations are detected in the Mutations are detected in the lacIlacI genegene

Color detection from Big Blue Color detection from Big Blue systemsystem

Results have been published in peer Results have been published in peer reviewed journalsreviewed journals

Characteristics of Characteristics of PfuPfu

High FidelityHigh Fidelity Native Hot Start activityNative Hot Start activity High ThermostabilityHigh Thermostability Slow polymerization rateSlow polymerization rate Available as native and cloned Available as native and cloned

enzymeenzyme

Accuracy of Polymerases Accuracy of Polymerases in PCRin PCR

Taq Tfl

100000

200000

300000

400000

500000

600000

700000

800000P

Taq Tfl

Extension TimeExtension TimePfuPfu requires 2 min/kb 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

Activity vs Temperature Activity vs Temperature PfuPfu and and TaqTaq

percent activity

25 35 50 60 75 95

Incubation Temperature (degrees C)

PfuPfu: Hot Start Every Cycle: Hot Start Every Cycle

M T TS ATG TMg Pfu

Polymerase Polymerase ThermostabilityThermostability

DNA Polymerase Polymerization Opt Half Life 95C Pfu 70-80C 19 hours

Taq 70-80C 1 hour

Characteristics of Characteristics of PfuTurboPfuTurbo

Improved version of Improved version of PfuPfu Same high fidelity as Same high fidelity as PfuPfu Higher PCR product yield thanHigher PCR product yield than Pfu Pfu

alonealone Can use 1 min/kb extension timesCan use 1 min/kb extension times Works well with low amounts of Works well with low amounts of

DNA templateDNA template

PCR Enhancing Factor PCR Enhancing Factor (PEF)(PEF)

Isolated from Isolated from Pyrococcus Pyrococcus Lacks DNA polymerase and Lacks DNA polymerase and

exonuclease activitiesexonuclease activities Information on identity/mechanism Information on identity/mechanism

of action is proprietaryof action is proprietary

PfuTurboPfuTurbo vs Other vs Other PolymerasesPolymerases

M PT P V DV Pw K T M PT P V DV Pw K T M

0.9 kb

5.2 kb

PfuTurboPfuTurbo vs vs PfuPfu

M T P T P T P T P T P

0.9 kb

1.9 kb

6.1 kb5.2 kb

Characteristics of Characteristics of TaqPlus PrecisionTaqPlus Precision

Blend of Blend of TaqTaq and and PfuPfu High YieldHigh Yield High FidelityHigh Fidelity LengthLength PCR Buffer PCR Buffer

– optimized for both yield and fidelityoptimized for both yield and fidelity

Accuracy of Polymerase Blends Accuracy of Polymerase Blends in PCRin PCR

020000400006000080000

100000120000140000160000180000

PCR with PCR with TaqPlus PrecisionTaqPlus Precision vs vs TaqTaq

Taq TaqPlus Precision M 2 5.2 5.4 10 15 M 2 5.2 5.4 10 15 kb

Characteristics of Characteristics of TaqPlus TaqPlus LongLong

Blend of Blend of TaqTaq and and PfuPfu Long targetsLong targets Fidelity similar to Fidelity similar to TaqTaq Two optimal buffers suppliedTwo optimal buffers supplied

– high salt buffer helps prevent high salt buffer helps prevent smearing in long target amplificationsmearing in long target amplification

– low salt buffer low salt buffer

PCR with PCR with TaqTaq vs vs TaqPlus TaqPlus LongLong

Qualities of Qualities of TaqTaq

Taq2000 Taq2000 is the recombinant is the recombinant TaqTaq Native Native TaqTaq is also available is also available TaqTaq is trusted by customers is trusted by customers TaqTaq has been well studied has been well studied

TaqTaqExtenderExtender

The The Pfu Pfu component of component of TaqPlus TaqPlus LongLong

Can be added to anyCan be added to any Taq Taq to make to make a polymerase blenda polymerase blend

Perfect Match PCR Perfect Match PCR EnhancerEnhancer Taq2000 native Taq

M - + - +

Opti-Prime PCR Optimization Opti-Prime PCR Optimization KitKit

12 buffers12 buffers– matrix of pH, Mg, KClmatrix of pH, Mg, KCl

AdjunctsAdjuncts– DMSO, formamide, BSA, glycerol, DMSO, formamide, BSA, glycerol,

ammonium sulfate, Perfect Matchammonium sulfate, Perfect Match Recommended for use with Recommended for use with TaqTaq

Related PCR productsRelated PCR products RoboCyclerRoboCycler

– gradientgradient Nucleotides and primers Nucleotides and primers StrataPrep PCR purification kitStrataPrep PCR purification kit Cloning VectorsCloning Vectors

– PCR-Script Kits PCR-Script Kits – Affinity protein expression kitAffinity protein expression kit

QuikChange site-directed mutagenesis kitQuikChange site-directed mutagenesis kit Cyclist DNA sequencing kitsCyclist DNA sequencing kits

PCR Related Products. Current Products n Thermostable DNA polymerases –Pfu –PfuTurbo –TaqPlus...

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