PCR: SOE, Bridge or Megaprimer?

[John Ladasky]

In article <boardman.3-0211981100390001 at path9.med.ohio-state.edu>,
Cindy Boardman <boardman.3 at osu.edu> wrote:
>In article <71difc$2ag at pmgm.Stanford.EDU>, jladasky at pmgm.Stanford.EDU
>(John Ladasky) wrote:
>
>>In article <boardman.3-3010981640170001 at path9.med.ohio-state.edu>,
>>Cindy Boardman <boardman.3 at osu.edu> wrote:
>>>Request for help:
>>>
>>>I am interested in expressing a chimeric protein and have been trying to
>>>create the junction between the two DNAs by PCR.  I have tried Bridge, SOE
>>>and Megapriming PCR and have had no luck.  Any generous and enlightened
>>>souls out there willing to save me?
>>
>>        I've made mutant proteins by PCR mutagenesis using the megaprimer
>>approach.  It should be essentially the same as making a chimera.  In my
>>experience the megaprimer works much better if it prepared to excess with
>>a blend of Taq and Pfu polymerase, and agarose gel purified.
>>
>>        Good luck!
>>
>>-- 
>>Rainforest laid low.
>>"Wake up and smell the ozone,"
>>Says man with chainsaw.                                 - John Ladasky
>
>
>
>Thank you for replying. I have tried using excessive amounts of gel
>purified Megaprimer, but amplified with Taq only, no Pfu.  However I would
>be surprised if this is the reason for my failure.  Im wondering if it
>isn't simply the design of my megaprimer that is the problem.  Could you
>tell me what size  megaprimers you have used, and how many annealing vs
>non-complementary bases did they typically contain. 
>
>Cindy

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