Pfu degrades primer from 5´end??

Michael T. MacDonell, Ph.D. visla at iix.netcom.com
Fri Nov 13 13:33:40 EST 1998


On Fri, 13 Nov 1998 11:35:44 +0100, "Georg Wille"
<moqch at mlucom6.urz.uni-halle.de> wrote:


>Though less than 100% coupling efficiency would mean some amount of shorter
>oligos, those should not generally be lacking the 5'-end but rather missing
>a random nucleotides within the sequence, as the coupling may continue after
>a "miss", no?
>
>Georg Wille
>
>
>

Dear George:

Nope. Just the opposite.
Coupling efficiencies of about 99.2% mean that, in the end, you have a
nested set of oligos, mostly full length (unless you are out to 70
bases or so), but with a collection of oligos shorter on the 5' end by

1, 2, 3, etc. bases.
During synthesis, when a particular DNA strand does not receive the
"next" base, the following step in synthesis acetylates (or caps) it.
This prevents internal deletions, since that strand can no longer be
reacted.
Now, having said that, there is a very small frequency of reversal of
the acetylated sites, so that in the aggregate, for a typical length
oligo, say 30-mer, there will be about a total of about 1% of the
oligos that have an internal deletion somewhere in the molecule, and
about 0.01% that will have two deletions. And so on.
Sudhir Agrawal has published on this problem. You can find his papers
if you are interested.  

Best Regards,
Mike MacDonell, Ph.D.
Chief Scientific Officer
Ransom Hill Bioscience




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