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pfu and Taq PCR mutation percentage

Gys de Jongh GysdeJongh at compuserve.com
Tue Jan 11 01:04:36 EST 2000


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. Start with N
DNA molecules of 3000bp. The number of right molecules after one cycle will
than be : N+(1-p_wrong)^3000*N. Because there were already N right molecules
and these molecules are duplicated in a proces where the chance for
duplicating the molecule right is (1-p_wrong)^3000. The total number of
molecules is just 2*N ; the wrong number is the difference between the two.
This process can be repeated for a number of PCR cycles.

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 = 21% (not 48 ?)
This number depends on the lenght of the PCR product and the number of
cycles.For 1000bp and 20 cycles %wrong molecules =8% For 1500bp and 35
cycles %wrong molecules = 19%

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

 --
Gys





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