Enzyme fidelity (Re: PCR mistakes)
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Wed Jun 3 09:40:35 EST 1998
Tim Fitzmaurice wrote:
> I went to look at Pfu, which everyone said round here was the best.
In terms of fidelity, Pfu really seem to be the best choice. We use the enzyme
very frequently, and have not encountered problems with errors.
> The company in question said that on 32 cycles and with a 2.5 kb fragment I
> could expect on average up to 100 errors.
100 errors in what? In one product molecule or in the total amplification mix?
I assume the company guy meant the latter. 100 errors in one product molecule
would make PCR technology useless for most approaches.
The error rate of Pfu is 1.3x10E-6 errors per base pair per duplication, as
compared to 8.0x10E-6 for Taq, or 55.3x10E-6 for UlTmA (data taken from the
Pfu manual; let´s assume they are roughly correct). So, Pfu is some 8fold more
accurate than Taq, and 35fold more than UlTma.
It is not easy to calculate the number of errors you have to expect in a given
molecule, because number of cycles does not say really how many duplications
you have. You can, however, ESTIMATE the number of duplications by comparing
template amount and product yield.
You use 10ng of (plasmid) template DNA, your DNA yield after PCR is 3ug. So,
your amplification is 300fold and the approximate number of effective
amplifications has been 8 to 9 (that´s 1->2->4->8->16->32->64->128->256->512).
Let´s say 10, as not all molecules are duplicated per cycle.
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