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pfu and Taq PCR mutation percentage

Gys de Jongh GysdeJongh at compuserve.com
Tue Jan 11 19:48:14 EST 2000


Gys de Jongh <GysdeJongh at compuserve.com> wrote in message
news:85eh2u$78b$1 at ssauraab-i-1.production.compuserve.com...
>
> Dr. Duncan Clark <Duncan at nospam.demon.co.uk> wrote in message
> news:9erByuAEgxd4EAXA at genesys.demon.co.uk...
> >
> > Mutation frequency for Taq is 8 x 10E-6/bp/duplication
> > Mutation frequency for Pfu is 1.3 x 10E-6/bp/duplication
>
> Should this be the number of wrongly incorporated bases after the
> duplication of one base (?) Is the dimension than 8x10E-6base/duplication
>
> >
> > So for a 20 cycle PCR of 3000bp target i.e. 1,000,000 fold
> > amplification, errors introduced by Taq are (8 x 10E-6) x (3000bp) x
> > (20) duplications or 48%.
>
> Can you explain this multiplication ?
> The outcome seems to be 0.48 ; why the 48% ?
> If you multiply the chance for incorporating one wrong base (8x10E-6) with
> the total number of bases than this will yield the number of wrong bases.
> However this will be 8x10E-6 x 3000bp x 2^20 (why 20 ?). If you want to
> express this as a fraction than you will ofcourse get the original 8x10E-6
> back (?)
>
> Here are a few other thoughts
> if the chance for incorporating a wrong base is p_wrong than the chance
for
> incorporating a right base is (1-p_wrong) The chance for duplicating the
> whole PCR product of 30000 bases is than (1-p_wrong)^3000 .To the power
3000
> because all bases of the PCR product must be right. I would not be
> interested in a PCR product with just one wrong base ; i would not care if
> it had more than one wrong base. I assume that the re-mutation of a wrong
> base in the right one in a following PCR cycle is neglectable.

I forgot about the double strand nature. Start with N ds DNA molecules of
3000bp. After melting there will be 2*N ss DNA molecules of 3000bases. Each
of those 2*N ss DNA molecules must be made ds by the polymerase. The number
of right ds DNA molecules of 3000bp will thus be : 2*N*(1-p_wrong)^3000.
Because the 2*N ss DNA are duplicated (made ds) in a proces where the change

for duplicating 1 ss DNA molecule of 3000 base completely right is
(1-p_wrong)^3000

> The total number of
>molecules is just 2*N ; the wrong number is the difference between the two.
  In a spreadsheet this process can be repeated for the next PCR cycle by
simply   considering the than actual number of  , just before calculated ,
completely right ds DNA molecules of 3000bp

>
> Lenght of PCR product=3000bp
> p_wrong=8E-06
> p_right=(1-8E-06)
   p_right for the product of 3000bp = (1-8E-06)^3000=0.976
   %wrong molecules after 20 cycles = 38% (not 48 ?)
> This number depends on the lenght of the PCR product and the number of
   cycles.For 1000bp and 20 cycles %wrong molecules =15% For 1500bp and 35
   cycles %wrong molecules = 34%
>
> I could mail the excel97 spreadsheet to anyone interested.
> I don't know the nummerical error propagation for excel in things like :
> (1-8E-06)^3000
However changing the 8E-06 to 7.95E-06 will yield other numbers.

 --
Gys






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