RE: Musing Daily Questions 🐮
There is a 2014 study which looked into the fidelity of 6 commonly used polymerases. Here is the link: https://www.hindawi.com/journals/mbi/2014/287430/
Important line from the summary: "Based on minimizing PCR errors, Pfu polymerase, Pwo polymerase, and Phusion all produce acceptably low levels of mutations."
Phusion created more indel mutations than Pfu and Pwo so it should not be used if you are doing an ORF cloning project.
This 2017 study (https://academic.oup.com/nar/article/24/18/3546/2359477) found that Pfu DNA polymerase achieved the best fidelity.
However, in the 2014 study, they found that "Aside from fidelity considerations, amplification efficiency values were significantly higher for Phusion and Pwo compared to Pfu, although further optimization of the PCR reaction for Pfu would likely improve efficiency values."
So likely you will want to choose Pfu or Pwo depending on your experiment.
Those are old polymerases though, there are several engineered polymerases which are vastly superior to those from Pyrococcus furiosus or Pyrococcus woesei. Phusion, as mentioned above is good as that is the combination of the polymerase/nuclease domains from an archaeal polymerase as well as a double stranded DNA binding domain, for increased affinity. So out of the above listed pols, for the use case I asked Phusion is hands down the winner.
However there are other commercially available polymerases like Kapa biosciences HiFi DNA polymerase, or New England Biolabs Q5 which both achieve superior fidelity to Phusion. Those two are polymerases which are used for Next Gen sequencing amplifications specifically because of their incredibly low error rate. In the case of Q5, its error rate (while one is reported) is more due to the detection of oxidative damage during DNA preparatory steps then it is actual mistakes made by the enzyme itself.
Were I to be amplifying a gene out of genomic DNA I would likely use Q5 or KapaHiFi DNA polymerases, as they would have the lowest error rate.
Oh, wow, really cool! Thanks for the lesson :)