Pfu polymerase in PCR

Victor Levenson levenson at uic.edu
Wed Feb 12 12:23:36 EST 1997


John Watson <watson_j at bms.com> wrote:

>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.



Have you tried increasing the amt of Pfu in the reaction? When I was
in a similar situation somebody suggested trying gradual increase in
Pfu concentration, and 2.5 fold (compared with Taq) increase did the
trick.
IMHO, increasing the time of 72^C step is unnecessary - when it works
Pfu will amplify your fragment within ~the same time as Taq.

Good luck,

Victor Levenson
Res Asst Prof
UIC, Dept of Genetics
levenson at uic.edu 
312-413-3887




More information about the Methods mailing list