Talk:Taq polymerase

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Latest comment: 16 years ago2 comments2 people in discussion

Quote: One of Taq polymerases' major drawbacks is its low replication fidelity. Without a 3' to 5' exonuclease proofreading mechanism to replace an accidental mismatch in the newly synthesized DNA strand, Taq polymerase cannot be used in experiments where an identical genetic sequence is required (such as in molecular cloning). End of quote

Actually Taq DNA polymerase can be used for cloning, usually it is just not worth the trouble and costs to use Pfu instead and one has to do a DNA sequencing of the vector plus DNA insert anyway. In all my dozens of cloning experiments I never had any trouble using Taq and never detected errors eventhough Taq has an error rate of 1 in 10,000.

Ph. D. in molecular biology

Indeed, over the past few months I've amplified up to 8 kb, and I've yet to seen my first mutation. I think it's a much exaggerated problem. 217.122.83.79 16:55, 24 April 2007 (UTC)Reply

Also, there are modified versions of taq that include proofreading activity, such as FastStart High Fidelity PCR System and Takara Ex Taq Polymerase. I've used both, and had great success with FastStart especially. I'm adding it to the article unless there are any objections.

That sounds all very well, but all of you failed to mention if you sequenced your PCR products before or after cloning. If you sequence your product directly after the amplification and before cloning into a vector, you're potentially dealing with a mixture of mutated DNA fragments, and only very close inspection of the sequencing traces will tell you if something is amiss (unless one mutation happened very early on in the PCR, you may only be able to see small "minor" peaks underneath large peaks). I've repeatedly used Takara's LA PCR Kit enzyme for long PCR and after cloning >5 kb PCR-amplified DNA fragments into bacterial vectors and then sequencing them, I (and others) nearly always found a mutation--that's even despite the fact that the Takara enzyme is supposed to have proofreading activity. So I'd be careful making these statements, unless you've given details on how you assessed potential mutations. Malljaja (talk) 11:30, 17 March 2008 (UTC)Reply

history edit

the taq polymerase was not isolated by Brock and Freeze. Those were the ones, who identified the termophilus aquaticus organism. As the citation rightly indicates, taq polymerase was isolated by Chien A, Edgar DB, Trela JM.

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