PCR: SOE, Bridge or Megaprimer?
boardman.3 at osu.edu
Mon Nov 2 18:14:57 EST 1998
In article <71l236$d3b at pmgm.Stanford.EDU>, jladasky at pmgm.Stanford.EDU
(John Ladasky) wrote:
>For my mutagenesis, I began by making a 90 bp megaprimer using two 20-mer
>oligos. There was a single mismatch with the original template ten bases
>from the 3' end of one primer. This megaprimer was then used with a third
>20-mer and the same template to create a 450 bp fragment.
>I should point out that I have also tried another way -- overlapping, four-
>primer PCR in order to create a point mutation. In this case the two tem-
>plates overlap only by 20 bases, which is perhaps more similar to your
>attempt to make a chimeric protein. This also worked, but I preferred the
>lower material costs of the megaprimer approach.
>The reason that I suggest adding some Pfu polymerase is that Taq polymerase
>is known to add nucleotides (usually a single A) that hang over the 3' end
>of a strand. Pfu polymerase (or another DNA polymerase with 3' -> 5' exo-
>nuclease acitivity) will trim off these ragged ends. If your megaprimer
>contains unwanted additional nucleotides at the extreme 3' end, the effici-
>ency of primer extension could be quite low.
>Rainforest laid low.
>"Wake up and smell the ozone,"
>Says man with chainsaw. - John Ladasky
The overlapping, four-primer approach is what I am currently trying.
(It's nice to know that it's worked for you at least.)
I had considered the problem of overhangs, and have tried mung bean
nuclease treatment of the products in order to cleave off any such extra
This also did not work......but it could be that my nuclease was no good.
I'll go ahead and buy some Pfu..it's worth a shot.
Thanks so much for your help.
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