Pfu polymerase in PCR

Dr. Peter Gegenheimer PGegen at
Fri Feb 7 19:08:27 EST 1997

In <5dcnh9$lnf at>, Fraser Dr N J <njf16154 at> writes:
>Bob Steinberg <RSTEINBE at ETOWAH.UOKHSC.EDU> wrote:
>>Does anyone have suggestions for making recalcitrant primer/template 
>>combinations work with Pfu polymerase. We are trying to amplify a short 
>>(about 120-bp) segment with very high fidelity-- eventually from genomic 
>>DNA, but presently from a plasmid template. We have no problem getting 
>>the correct product with Taq or ULTma polymerases, but get nothing with 
>>Pfu, even though we have dropped the annealing temperature by more than 
>>10 degrees from what works with the other polymerases and have 
>>phosphorothioate linkages for the three nucleotides at the 3'-ends of our 
>>primers. Extension times at 72 degrees C have been 2.5 min, which should 
>>be more than generous for the short piece we are trying to amplify. We 
>>have tried two lots each of Pfu polymerase, Pfu buffer, and dNTPs. Has 
>>anyone looked at varying salts and/or magnesium with Pfu?

Pfu is a proof-reading polymerase, that is, it has a 3'--5' exonuclease activity, as does VENT polymerase (N.E. Biolabs). This exo activity can degrade your product DNA (as well as the primers). For that reason, NEB recommends an extension time of NO GREATER than 1 min per kilobase, or 0.12 min for your 120-mer. If the annealing temp is close to the elongation temp, try reducing  the annealing time to essentially none. 
The optimal annealing temperature should not vary with polymerase (unless it is clse to the extension temp). 

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