Pfu polymerase in PCR

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Mon Feb 10 11:23:40 EST 1997


In article <5dgg5r$5fr at raven.cc.ukans.edu>, PGegen at UKans.edu   (Dr. Peter
Gegenheimer) wrote:

> In <5dcnh9$lnf at ukwsv3.ggr.co.uk>, Fraser Dr N J <njf16154 at ggr.co.uk> 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). 
> 

Pfu is a proofreading POLYMERASE. It will NOT degrade your DNA in presence
of nucleotide triphosphates (this is why you should add the polymerase
last). Only after misincorporation of a nucleotide, Pfu will remove this
nucleotide and then continue polymerizing. Use 2min/kb or more (dependent
on your cycler, titrate). There are, however, some sequences that Pfu
polymerase refuses to make. In my hands, all these sequences have turned
out to be G-rich (anyone to confirm this?).

hope this helps
Frank

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



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