yatsen at wam.umd.edu
Thu Dec 21 20:11:22 EST 1995
On Thu, 21 Dec 1995, Ned Mantei wrote:
> 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.
It may not be true.
It is probably true that errors in Cycle sequencing will not affect the
final sequencing result of a pool of template samples. However, the
error in initial PCR reaction could show up IF the starting material is
minimal. Errors in early amplification cycles can accumulate and generate a
pool of templates as the final PCR product. It is just like statistical
analysis: the average is meanless if the sample number is not big enough.
But, I agree that sequencing the PCR product directly will have much
better chance in reviewing the real sequence.
>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
Finally, high fidelity Pol may reduce the error rates to a point. But,
the final answer to this kind of question is to compare your sequence to
the genomic sequence.
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