Pfu degrades primer from 5´end??
gkidd at aptagen.com
Thu Nov 12 20:12:39 EST 1998
Yes, it is possible that your primers have a high content of incompletely
synthesized primers. The coupling efficiency is usually 98-99% for each
reaction in the synthesis of an oligo, if you're dealing with a good company.
This means that for a 20-mer, only about 67-82% of the product will be
full-length. Since chemical synthesis occurs in the 3'-5' direction, the
incomplete oligos will be missing the 5' end. Regarding your observation that
different PCR conditions seem to affect the severity of the problem, this may be
due to the influence of the various PCR conditions on the ability of the shorter
oligos to bind to the template. Good luck!
Geoffrey Kidd, Ph.D.
Technical Services Manager
Torsten Börchers wrote:
> 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
> Any comment is highly wellcome
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