Pfu degrades primer from 5´end??

Torsten Börchers borcher at uni-muenster.de
Thu Nov 12 09:29:19 EST 1998


Dear colleagues,

I am aware of earlier discussions on this list about the 3´-5´ exonuclease
activity of Pfu polymerase and the impact on PCR conditions.

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).

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

Any comment is highly wellcome

Torsten

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