mbi050 at abdn.ac.uk
Fri Jan 12 09:20:16 EST 1996
Ned Mantei (mantei at neuro.biol.ethz.ch) 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. 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
I agree with this comment. There numerous people who say PCR based sequnecing
is flawed but, normally have no experience of it themselves.
When I, or others I know have done PCR to get a product then PCR based cycle
sequencing and compared it to know sequence from radiolable standardDD-methods
we have had no problem.
All our the screening of a gene for say random point mutations can easily be
done by using overlapping PCR products of 400 bp, each amplified twice
independantly, and then sequence on both strands. With PCR and automation
this takes less than a day to do, and then overnight to read of the sequence.
If you then get a mutation or mismatch you can see it from two independant
amplifications and both strands, in practice we normally end up with
this being in triplicate.
In the sequecing of a 1.6 kb ORF I've seen only one miss-base in one from PCR
product, which was a clear as day.
As for automated sequencing the same is true, if it was as bad as some people
make out then nothing would ever get sequenced.
given the speed and the, enormous excess of primer that you normally make
its no problem repeating samples or doingduplicates etc.. to ensure acuracy!
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