Error rate in Taq pol. 1:270?

Dr. Duncan Clark duncan at
Mon Feb 10 04:30:28 EST 1997

In article <5dlh6s$q02 at>, derk at writes
>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.

The error rate depends a lot on the no. of cycles you do. More cycles
more errors. What did you amplify the fragment from - a cDNA clone? If
so you could use 1-10ng of clone and say 10 cycles maximum. You can
destroy the starting template with Dpn I or alkali denature it first. If
you are going from mRNA/reverse transcriptase then it may be the RT that
is causing the errors and not the PCR enzyme. For the best error rate
you have to have some proof-reading enzyme present. Finally have you
considered that there could be a sequence error in the original cDNA
sequence and your PCR result is actually correct? 


The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Dr. Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288

More information about the Methods mailing list