Error rate in Taq pol. 1:270?

Tom Chappell t.chappell at ucl.ac.uk
Mon Feb 10 09:32:26 EST 1997


In article <5dlh6s$q02 at mserv1.dl.ac.uk>, <derk at hubrecht.nl> wrote:

>Hello all,
>
>To make an overexpression construct I amplified an 810 bp cDNA fragment and
>subcloned it. Upon sequencing it turned out to have 3 nucleotide
>substitutions, one c to t, one g to t and one t to a. This leads to an error
>rate of 1 per 270. To me this seems abnormal high. What are others
>experiences? How can I improve the fidelity of the amplification, except for
>using expensive high fidelity polymerases? I used GoldStar DNA polymerase
>from Eurogentec and an Idaho Technologies air thermal cycler.
>
>Thanks for your consideration.
>
>Derk ten Berge
>
>Netherlands Institute for Developmental biology
>Utrecht, The Netherlands
>derk at hubrecht.nl

You're using faulty logic to calculate the error rate. If you've done a 30
cycle PCR, the polymerase has synthesized more than 24,000 bases to
generate the single DNA fragment that you've subcloned and sequenced. Your
error rate would be about 1 in 8,000, which isn't too bad for Taq. I
typically sequence 6 independent isolates if I have no functional assay
for fidelity. Errors tend to be independent, indicating that they've
occurred sometime during the PCR reaction other than in the first few
cycles. I've only once gotten an error that was present in 5 of 6
independent isolates.

Tom Chappell
MRC Laboratory for Molecular Cell Biology




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