Hello. I’m planning to use overlap extension PCR for DNA insertion into a vector, as it is explained by Briskin and Matsmura in “Overlap Extension PCR Cloning”. I have a question about the polymerase since in the articles they prove different polymerases for the experiment and they conclude that Phusion polymerase is better for the procedure. I don’t have that polymerase at my lab, but I have a taq polymerase, which is the difference between this two? Have someone done something similar (Overlap extension PCR) using a taq-polymerase? Why using Phusion polymerase?
I attached the link of this protocol if someone wants to read it:
http://www.biotechniques.com/BiotechniquesJournal/2010/June/Overlap-extension-PCR-cloning-a-simple-and-reliable-way-to-create-recombinant-plasmids/biotechniques-280116.html
A few reasons:
I'm guessing Taq would probably work with a small vector (ie pBluescript) and a small insert, but overlap-extension PCR is already a very unpredictable process. If you can get Phusion or Q5 it would probably be worth it though to save some hassle, I don't think they're particularly expensive.
We have done this in my lab using Pfu polymerase. That works for this reaction, and with less error rate and higher fidelity than Taq polymerase.
Yes, as Alexandra Johnson and Chitra Kannabiran mentioned, for cloning, better use enzymes with proofreading activity (Phusion or Pfu or others - they are more expensive than Taq the cheapest of all polymerases). Taq, one better uses for genotyping and sequencing, after which there is no downstream processing of the pcr products.
However, after successful insertion, you have to prove correctness of your construct by sequencing, because you don't want to go on with a construct bearing a mutation (putting all your following research in question). And even if the inserted fragment has no mutation, the ligation sites could bear some mutations ...
The speciality of Phusion is that it is fused to a DNA-binding protein's domain (that's why its name is "Phusion"), so that it stays attached to the template DNA over a longer distance than usual polymerases. Therefore, Phusion is especially suited to do long-range PCR (PCR products of several kilobases of length). To profit from the higher accuracy of this protein, the slightly lower extension temperature of 68°C is crucial (Taq's extension temperature is 72°C). I used Phusion extensively to generate > 10 kb longrange products during my PhD thesis - in the last years, I used only Phusion for all my cloning stuff. (Buy several packs and get discount from provider, then it becomes cheaper per unit. And Taq is sometimes not much cheaper ... Especially, you save sometimes a lot of time when using Phusion). You can contact me personally for an improved protocol if you want.
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