High Fidelity DNA Polymerase: how many mutations per every kb of amplified DNA o - (Oct/17/2005 )

Hi,

I talked to my co-worker the other day about new tricks in cloning and what he told me was really astonishing :

When making a transgenic mouse model, he PCRed a 2 kb fragment of DNA with High Fidelity Taq Polymerase (Roche) to use it in his cloning strategy..... later, he sequenced the amplified product to find NO MUTATIons whatsoever....

I think it is a nice topic for the forum:

How far can one go with high fidelity DNA polymerase? Does anyone have any experience with the reliable amplification of 3 kb fragments? 4 kb?

thanks

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I have cloned and sequenced PCR products of 3 or 4 kb many times with no errors, using high fidelity polymerase...

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hi
i've sequenced a clone of 13kb and only one mutation occured.
Btw, pol are now proofreading and you can expect easily 5kb without any errors.

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I've used the same polymerase for a 2,2 kb amplification. Checked 2 clones, 1 had a mutation, the other didn't. So it's a matter of luck in choosing the clones.

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Guuuyyysssssss!!!!

that's really cool! 3-4 kb and no mutations...we're talking DNA polymerase from Roche, right? It would so help me with my cloning...I 'd rather seq a bunch of clones rather than wasting time of doing the timeless RDs and gel isolations....

thanks a ton!!!!!!!

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By "from Roche", do you mean purchased from them, or Taq-based? I used Invtrogen's HiFi polymerase (see here), but it's a mixture of Taq and another enzyme...

Any of the Hi Fi thermostable polymerases (e.g. Pfu from Pyrococcus furiosus, Vent (aka Tli) from Thermococcus litoralis, etc.) should work fine...

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Hi HomeBrew,

I mean the Expand High Fidelity PCR System, Cat # 1 732 641, that is sold by Roche Applied Science. It is composed of an enzyme mix containing thermostable Taq DNA polymerase and Tgo DNA polymerase. They mention that this system can efficiently amplify DNA fragments up tp 5 KB. On the other hand, they don't say a word of the number of possible mutations when one amplifies such a fragment.....

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Most hifi enzymes are indeed mixes of taq and a proofreading polymerase (such as Tgo). They have a higher fidelity than taq and are good at amplifying longer fragments.

These mixes have however a lower fidelity than pure proofreading enzymes, such as the ones homebrew mentioned. But, even with these enzymes, you can never guarantee that your clones have no mutations at all. You should always check because no polymerase whatsoever has a zero error rate.

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I think the Expand High Fidelity PCR System from Roche should work fine for you. The success of your cloning (success being defined as capturing a clone with no sequence errors) is always subject to the two very true points made by vairus -- that no Taq mixture is as good as a pure high fidelity polymerase, and that there is no such thing as a polymerase that is error proof (a good thing for evolution, BTW).

So, you should sequence your clones before proceeding, to insure you're working with an unaltered copy of the DNA of interest...

I sometimes perform several separate transformations, and pick colonies from each, to assure that each clone is produced by a separate event, and that my clones are thus not siblings of one another. Luckily, I've never found a sequence error in the dozen or so times I've cloned genes >3 kb using PCR.

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I have no problems with my 5-6kb products using Triple master from Eppendorf. For what I remember none of my clones to date had mutations.
Clarice

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