Pfu degrades primer from 5´end??
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Nov 12 09:55:15 EST 1998
In article <003301be0e48$82fff800$03fdb080 at arnheim.uni-muenster.de>,
Torsten Börchers <borcher at uni-muenster.de> 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....
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