Primer deletion

The Great American Gene Company geneco at ix.netcom.com
Fri Nov 29 12:21:26 EST 1996


We see this also, from time to time.  I can tell you that it is virtually
impossible, from a standpoint of primer synthesis, for a synthesizer to
effect the deletion of even one base.  The reason is that all modern
synthesizers employ a strategy wherein acetylation follows the coupling
step.  This means that when the "next" base is added to the growing
oligonucleotide chain, those sites that do not get the "next" base (the
reaction is not quantitative), are capped.  This is done specifically
to prevent any oligo from inadvertantly containing a deletion.

Sometimes the acetyl cap does reverse, and it is common that a percent
or so of the final oligo yield will contain single base deletions.  It
is inconceivable, however, that you would find a several-base deletion,
and then find it consistently in several copies of the oligo.

I hope that others who have some experience with the molecular biology
side of this phenomenon (rather than the synthesis side) will
contribute to this discussion.  It has plagued folks on both sides of
the problem for some time.

Best Regards!
Mike MacDonell, Ph.D.

In <329F555A.77A8 at mail.ncku.edu.tw> usrer <user at mail.ncku.edu.tw>
writes: 
>
>Dear Netters,
> I'm working in doing mutation by inverse PCR. When I got the
sequence,
>I found there're serious deletion between the tail-tail region of the
>primer couple. I used two polymerase (Taq from promega,2U; pFU 1/32 U
in
>25microlitter) and I've tried various buffer soln.(even from barner
>LA-buffer) , but I still got 2-5 based deletion. 
>I've wondered whether there're problem about Taq, and then I switched
to
>use pFU only to amplify two fragment respectively with additional two
>primer. That's to say I amplified the N- & C- fragment of my gene by
>pFU, 
>then ligased them (blund ligation). However, I got a 5 bases deletion
>mutant again.
>Would anyone can tell me whether the problem is due to my method or
the
>primer?

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