Bp-exchange by PCR
vvsvetlov at utmem1.utmem.edu
Tue Dec 10 08:35:23 EST 1996
In article <32AD2B0D.3E5D at urz.uni-heidelberg.de>,
klaus.lun at urz.uni-heidelberg.de wrote:
> I have amplified a 1.2 kb fragment with Pfu Polymerase and after
> sequencing I found 2 Bp-exchanges, leading to aminoacid substitutions.
> So I was wondering if this is the 'normal' mutation rate you get with
> Pfu-Polymerase; because I want to express this fragment I absolutely
> need no Bp-exchange. So how many PCR-poducts should I clone ? 10, 20 ? I
> perform the PCR with 30 cycles ..
> So please, help me ..........
In my experience with Pfu/Pwo polymerases the rate you've got is too much
unless you picked a mutated clone out of 10 wt. The number of cycles of
course is important but that's not all that is. First you can increase the
amount of template and reduce the number of cycles. Get the annealing
temp. as high as you can and reduce the extension time (if it is more than
10 sec), another thing that reduces the rate of misincorporation is the
drop in the concentration of dNTPs. BTW what's your template?
Another thing is that I found Pfu more sensitive to all kinds of crap
present in template or oligo preps than Pwo. I've got some primer/template
pairs that don't work with Pfu but work nicely with Pwo, it is never the
other way around. I've done some amplifications with Pwo up to 3 kb long
w/out significant misincorporations using low dNTPs and rapid ramping
cycles. The rationale for doing it rapidly (at the times close to kinetic
optimum) is that when polymerase misincorporates it's often results in or
from stalling, during quick cycling such stalled complexes don't have time
to extend and form an abortive/non-amplifiable products so that
misincorporations don't accumulate.
Another thing - just to be on the safe side check whether you are using
Pfu alone or Pfu/Taq mix (sometimes used for extralong amplifications).
Hope this helps.
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