Pfu degrades primer from 5´end??

Dr. Duncan Clark Duncan at
Thu Nov 12 09:55:15 EST 1998

In article <003301be0e48$82fff800$03fdb080 at>,
Torsten Börchers <borcher at> writes
>However, we upon sequencing or restriction analysis consistently observe PCR
>products (cloned into a vector) in which nucleotides (as much as 6 or more)
>are missing at the end. Our suspicion is that Pfu also degrades primers from
>the 5´end. Since we do not often sequence Taq generated cloned PCR products
>I cannot really say this is Pfu specific, but we feel that the problem
>became less severe when strictly following the recommendations for Pfu (i.e.
>enzyme at last, ice, hot start).

How do you know that this is a function of the Pfu and not say an
'artifact' of the subsequent cloning unless you are directly sequencing
the actual PCR product, into the primer region. Pfu definitely has no
5-3 exo activity due to it not having the appropriate catalytic
residues/conserved domains necessary for this activity. However there is
nothing to stop an 5-3 exo activity being a contaminant in your enzyme
batch or say the ligase or RE's used for cloning etc

>Has somebody else observed this phenomen or has a reference on hand.
>Alternatively, is it possible that we simply see imcompletely synthesized

I'd say it is unlikely to be the primer unless you have very poor
coupling efficiency during the synthesis leading to only a small
fraction of the finished oligo being of correct length. 

The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382

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