Talk:Taq polymerase

From Wikipedia, the free encyclopedia

	This article is part of WikiProject Genetics, an attempt to build a comprehensive and detailed guide to genetics on Wikipedia. If you would like to participate, you can edit this page, or visit the project page to join the project and/or contribute to the discussion.
???	This article has not yet received a rating on the quality scale.
???	This article has not yet received an importance rating.

	MCB Portal This article is within the scope of the Molecular and Cellular Biology WikiProject. To participate, visit the WikiProject for more information. The WikiProject's current monthly collaboration is focused on improving Restriction enzyme.
Start	This article has been rated as Start-Class on the assessment scale.
High	This article is on a subject of High-importance within molecular and cellular biology.
Article Grading: The article has been rated for quality and/or importance but has no comments yet. If appropriate, please review the article and then leave comments here to identify the strengths and weaknesses of the article and what work it will need.

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)

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)