Pfu polymerase mixed with Taq polymerase

Hi there,

Have heared that one may mix Pfu and Taq polymerases (Fermantas). Taq
is copying Pfu verifying. Have any of u tried this combbination? I'm
planning to introduce two nucleotide changes in 1kb DNA fragment. So
far I made set of 6 PCRs (I'm using so called SOE-PCR method with
overlapping extensions) but my RedTaq polymerase made so many mistakes
that I started to sworn it very badly.

Thanks for advise

I come from a country where less that 1% of country earnings is
subscribed for science. We do not have money for reactions using only
Pfu polymerase, so that's why I asking u these things


Jacob

That won't work. Pfu is a polymerase. It's not capable of
proofreading without polymerizing. So, it won't follow Taq around and
act as a high fidelity proofreader. If you mix the two enzymes, you'll
have some strands of DNA that are high fidelity (which were synthesized
by Pfu), and other strands that will be low fidelity (which were
synthesized by Taq). The only solution here, as I see it, is to only
use Pfu if you are needing high fidelity reactions.

--Alex

************************
Alex B. Berezow, Grad Student
Dept. of Microbiology
University of Washington School of Medicine
Seattle, WA 98195

<[Only registered users see links. ]> wrote:



That works since some companies are selling those kind of mix (I think
it is BD, but I can't remember for sure) mostly for long PCR. You are
supposed to have speed (Taq) and accuracy (Pfu). But I have no idea how
it actually works. And you'll have to be carefull with buffers.

--
Patrick

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.

[Only registered users see links. ]

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.

--Alex

***************************
Alex B. Berezow, Grad Student
Dept. of Microbiology
University of Washington School of Medicine
Seattle, WA 98195

Thanks for advice. I've tried Pfu:Taq in 1:10 proportion. In agarose it
looks great - comparing to my PCRs when I was using RedTaq (Sigma) I
have much more product (without cjangieng PCR program, 25 cyceles only)
I'm thinking if may be rhis result is even too good, cause more product
- more mistakes - even when I use high fidelity polymerase, am I right?

Soon I will sequence my products and we will see.

Jacob

On 28 May 2006 00:42:26 -0700, [Only registered users see links. ] wrote:



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

Bob wrote:

Your explanation seems quite logical to me.

As for the course on Black Magic....I do believe I've taken that. We
weren't allowed to ask any questions, either.

--Alex

************************
Alex B. Berezow, Grad Student
Dept. of Microbiology
University of Washington School of Medicine
Seattle, WA 98195