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PCR fidelity

Ned Mantei mantei at neuro.biol.ethz.ch
Thu Dec 21 01:43:23 EST 1995


In article <4b9cu7$i26 at thorn.cc.usm.edu>, rbateman at ocean.st.usm.edu
(Robert Bateman) wrote:

> I have a graduate student who is using RT-PCR to amplify a large section 
> of the coding region (75%) of a peptide processing enzyme from several 
> mammalian species. We intend to sequence these and compare the sequences 
> to identify conserved residues. His committee is concerned about the 
> fidelity of Taq polymerase even though he is directly sequencing the PCR 
> products. 


If he is *directly* sequencing the PCR products, without cloning them,
then he is reading the *average* sequence. The 0.1% or less errors at each
position never show up. There would only be a problem if the polymerase
always made a mistake at >25% or so frequency at one particular position,
something for which there is no evidence at all. The problems with
mistaken incorporation arise when one looks at individual clones. I don't
think the committee understands the problem.

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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