Error rate in Taq pol. 1:270?

Dr. Duncan Clark duncan at genesys.demon.co.uk
Mon Feb 10 04:30:28 EST 1997


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

Duncan   

--------------------------------------------------------------------------------
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
http://www.dnamp.com
http://www.genesys.demon.co.uk



More information about the Methods mailing list