IUBio

[Microbiology] Re: Pfu polymerase mixed with Taq polymerase

Bob bbx107.XYZ at excite.XYZ.com
Sun May 28 12:39:09 EST 2006


On 28 May 2006 00:42:26 -0700, biovirus04 at gmail.com wrote:

>Well, I'll be the first to admit when I'm wrong.  It looks like Pat is
>right.  Another thread (on another website) discusses this very same
>issue.
>
>http://www.protocol-online.org/archive/posts/12810.html
>
>I have no idea how or why this protocol would work, but what do I know?
> Someone on here was using a 10:1 ratio of Taq to Pfu.....so give it a
>shot.
>
>This protocol is very counter-intuitive (at least to me) on,
>mechanistically, how these enzymes would cooperate to achieve a higher
>fidelity reaction.  Oh well.
>


I'd take it as more empirical than theoretical. But in that spirit,
here is a rationalization (rather than an explanation). 

Processivity (ability of the polymerase to proceed down a  chain
without falling off) is not absolute. The Taq Pol is used here without
any processivity apparatus (e.g., beta clamp). Stalling at an error
(mismatch) enhances falling off. So if Taq makes a mistake and falls
off, the proofreading Pol may bind and correct the error. 

I vaguely recall when this came out, but don't remember any details. I
would assume that the procedure itself is strictly empirical. It
should be covered in your course on Black Magic. 

I'd suggest that the OP follow the (Pfu) supplier's recommendations.

bob




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