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I was reading a manual for kit that requires you to amplify DNA by PCR before analyzing it in the kit. The manual suggests mixing proofreading and non-proofreading polymerases:

For PCR products >2500 bp in length, use a DNA polymerase blend containing Taq DNA polymerase supplemented with a proofreading DNA polymerase

How would this improve the amplification of long products? It suggests using only a proofreading polymerase for fragments below 2500 bases. Does the addition of a non-proofreading polymerase improve speed? I have TAQ and PFU Ultra, but both instructions say to use an extension time of 1 minute per kb.

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The Taq polymerase is a rather fast enzyme (the synthesis rate is around 1 kb/min), but the backdraw is that it has no 3'- 5'exonuclease activity which would enable to proofread the newly synthesized DNA. This leads to the introduction of errors and the chance of getting these is higher the longer the DNA you want to synthesize is.

Polymerases which are able to proofread (which have the exonuclease activity) like Pfu have a much lower error rate, but are on the other hand also much slower, typically around 0,5 kb/min. Pfu is also more expensive than the Taq polymerase. See table 1 from reference 1 below:

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To combine the advantages of both polymerases, you typically use a blend of Taq and Pfu. Then you get the high processivity of the Taq combined with the proofreading capacity of the Pfu and get good yields of DNA with a low amount of mutations. See also reference 2 for details on this system.

References:

  1. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase
  2. Effective amplification of long targets from cloned inserts and human genomic DNA.
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  • $\begingroup$ To add to the answer, the reason this works is because Taq will stall and fall off the DNA after a misincorporation. (Taq isn't particularly progressive.) If Taq just continued on after misincorporation, Pfu wouldn't have a chance to edit it. By falling off, Taq gives the slower Pfu time to bind to the DNA, edit out the misincorporation, and continue on. When there's no Pfu, Taq does eventually continue on past the misincorporation. But that process tends to be slower than that for Pfu binding and editing, so when Pfu is present the error correcting path is preferred. $\endgroup$ – R.M. Jun 1 '16 at 14:49
  • $\begingroup$ So I've been having trouble with another project, which requires I use PCR to amplify a 4400 base gene, or an entire plasmid. I've been using PFU ultra alone, and the reaction takes 5 - 6 hours. How much Taq polymerase should I add to improve the reaction speed? $\endgroup$ – user137 Jun 2 '16 at 0:44

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