Polymerase error rate as a function of temperature?

Duncan Clark via methods%40net.bio.net (by blackhole from abuse.plus.com)
Wed Aug 18 05:08:55 EST 2010


Historians believe that in newspost <i4eidq$g8q$1 from news.albasani.net> on 
Tue, 17 Aug 2010, DK <dk from noemail.thankstospam.net> penned the following 
literary masterpiece:
>I would like to do everything humanly possible to reduce the
>frequency of PCR errors in some reactions. I use PfuUltra
>Fusion, which has probably highest fidelity of them all.

Pfu with their modified Sso7d domain plus Pfu pol- enzyme to give extra 
proof-reading exo activity. Also dUTPase as well?

>
>It also is very processive. I am wondering if reducing
>an extension temperature (say, to 65C) will reduce not
>only processivity

I would expect the processivity to stay the same regardless of 
temperature and just the extension rate to be slower. Although dropping 
from 72 to 65 will have negligible effect on the rate.

>but also an error rate. Or will the error
>rate remain the same?

Again I would expect to stay the same.

>
>I guess the answer depend on the precise mechanism
>of polymerization errors (e.g., which are the ones that
>escape proofreading corrections?) - and I just know know
>what it is.

Maybe Wayne Barnes or one the Stratagene people have published what the 
standard error format is for Pfu

Aha.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146123/.

Other than keeping the cycles as low as possible and lots of initial 
template there's not too much more you can do.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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