It is well known that the first DNA polymerase, Taq, is quite error prone. Newer generation commercial enzymes that have either been isolated from different thermophile species or have been improved by recombination are less error prone. How are these error rates compared? For instance, if this is done by Sanger sequencing, the average signal will dominate in reading the output and so it will be very unlikely to pick up on errors by this method.

  • $\begingroup$ The Taq products could be cloned before sequencing. It's still complicated though, because the same error in two products could represent the same error arising de novo twice, or the propagation of a single error that arose in an early round of amplification. $\endgroup$ – Alan Boyd Sep 12 '12 at 7:04
  • $\begingroup$ This is why I was sceptical about a sequencing-based approach. I suppose the method described by Alan Boyd below is a functional approach, but this still masks synonymous mutations and therefore under-represents the true error rate. $\endgroup$ – user560 Sep 12 '12 at 22:24

According to their website New England Biolabs use a version of the approach pioneered by Wayne Barnes, as described in:

Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147

This is basically an assay for the mutation rate in a PCR-amplified lacZ (β-galactosidase) gene, assayed by transforming E. coli, plating on the chromogenic β-galactosidase substrate Xgal, and then scoring white colonies as mutated genes. Also according to NEB, Agilent Technologies use a similar mutational assay, but based upon the lacI (lac repressor) gene.

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    $\begingroup$ Thank you for the reference. This seems like the most pragmatic approach to this problem. Even though by design this method under-reports the error rate, it would be a consistent bias across polymerases. $\endgroup$ – user560 Sep 12 '12 at 22:25
  • $\begingroup$ It's possible that they have calibrated the mutational assay to a more rigorous approach somewhere along the line, but I'm just guessing. As you say, for them the relative fidelity is more important than the absolute value. $\endgroup$ – Alan Boyd Sep 13 '12 at 9:56

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