Pfu/Taq in PCR reactions

Christopher Paul Palmer sasplmrc at reading.ac.uk
Fri Mar 3 09:52:09 EST 1995



On Thu, 2 Mar 1995, Paul N Hengen wrote:

> On 26 Feb 1995 jnewton at molbiol.ox.ac.uk wrote:
>  
> > ...I've seen a number of 
> > people advocate mixing Taq DNA polymerase with Pfu DNA polymerase in order to 
> > improve/reduce the rate of mismatched base incorperation during PCR, however, 
> > I can't understand why this should help.
>  
> ...and on 28 Feb 1995 segdoh at uxa.cso.uiuc.edu wrote: 
>  
> | The reason some PCR kits have Pfu and Taq mixed together id to reduce the
> | error rate (Pfu) and to facilitate TA cloning (Taq).  Pfu does not leave
> | nontemplate terminal A overhangs like Taq which are required for TA cloning.
>  
> Hold on! Now I'm confused. Is it better for TA cloning to have a mixture of
> polymerases including Pfu which will polish the A's off the 3' end, or
> NOT to have Pfu ...allowing Taq to add the A's to the 3' end of the PCR product?
>  
> *******************************************************************************
> * Paul N. Hengen, Ph.D.                           /--------------------------/*
> * National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
> * Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
> * Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
> * Frederick, Maryland 21702-1201 USA              /--------------------------/*
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> 
	Since Pfu polymerase has proof reading activity it has a lower 
rate of mis-matched incorporations, an obvious advantage over Taq which 
has no such activity.  However since Pfu doesent possess a Terminal 
transferase activity it is difficult to clone Pfu PCR products into TA 
vectors.  However this can be circumvented by performing the PCR with Pfu 
initially, following PCR completion, Taq polymerase (1-2 units/100 ul) 
can be 
added to the PCR mix which is subsequently incubated at 74C for 10 
mins.  As a result the PCR product is amplified with a lower rate of misincor
porations and also has A residues transferred to the ends of the PCR product by

 the terminal transferase activity of Taq and thus should be easily clonable
into TA 
vectors.  Another factor which can effect TA cloning is the 
concentration of dATP in the reaction mixture, which may be depleted 
after PCR amplification, therfore it may be advisable to add fresh dATP 
to the reaction mixture after PCR amplification and before adding Taq 
and incubating at 74C for 10 mins.  > 


Chris



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