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pcr taq pfuu

Douglas Rhoads DRHOADS at MERCURY.UARK.EDU
Fri Feb 3 09:40:22 EST 1995


> To:            biochrom at net.bio.net
> From:          frog <pennie at access2.digex.net>
> Subject:       Re: pcr taq pfuu
> Date:          Thu, 2 Feb 1995 13:03:22 -0500

> 
> 
> On Thu, 2 Feb 1995, Philip L. Carl wrote:
> 
> > Presumably it does get back on and proceed some considerable fraction of
> > the time-that's why Taq PCR is error prone.  The key here is the ratio of
> > exo activity to polymerase activity, and while I agree the ratio of 100:1
> > is a bit surprising, it's not so surprising that I find it a mystery.
> > 
> > Phil Carl
> 
> 
> I agree but there are other factors which could come into play;
> 
> 1) How far does taq continue after introducing an error. Does it fall off 
> right away or not? If it can proceed further than pfu's ability to 
> proofread back then a significant number of errors may go undetected.
> 
> 2) What is the relative affinity of taq vs pfu for a missmatched 
> template/primer. In other words if taq is poorer (say 100 times less 
> affinity than pfu)  at settling onto a mismatched elongating template 
> you can imagine it falling on and off without getting anywhere. Then 
> along comes the 1 in 100 molecule of pfu with no such distaste for a 
> mismatched base pair and off it goes.
> 
> If anyone can elaborate on any of these points I'd be happy to know 
> (eg relative Km values for correct and missmatched templates, limit of 
> "back" proofreading of pfu, processivity of taq after error). 
> 
> Later.........
> 
> Frog.

You might check out a paper by Huang, Arnheim and Goodman in Nucleic 
Acids Research 20(17):4567-4573. `Extension of base mispairs by Taq 
DNA polymerase: implications for single nucleotide discrimination in 
PCR.

They don't compare Taq and Pfu but they do sort out some of your 
association/dissociation questions for Taq.  Maybe that will help, 
maybe not.
 
 
//========================================================\\
||Doug Rhoads              || Dept. of Biological Sciences||
||drhoads at mercury.uark.edu || 601 Science Engineering     ||
||drhoads at uafsysb.uark.edu || University of Arkansas      ||
||501-575-3251             || Fayetteville, AR 72701      ||
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||     My Dogma Just Got Run Over by Someone's Karma      ||
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