Pfu polymerase in PCR

John Watson watson_j at bms.com
Fri Feb 7 16:41:27 EST 1997


Bob Steinberg 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?

Before killing myself making Pfu work on a stubborn template, I'd try some other 
enzymes or enzyme combinations.  We have had good luck recently with the EXPAND 
enzyme mix from Boehringer (a mix of Taq and Pwo polymerases).  Not as high 
fidelity as Pfu, but better than Taq alone (1 ~1500 bp clone out of 3 sequenced 
had the correct sequence) and much more robust than Pfu -- we couldn't get Pfu to 
amplify this sequence at all.

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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