PCR fidelity

Chris Greene cgreene at bimcore.emory.edu
Wed Jan 3 15:13:55 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

Even with direct sequencing of pcr products, I think that combining two or more independent 
reactions is prudent.  There is always the chance that the error will occur early in the 
amplification series, and become a major component of the final amplification products.  --

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