Taq polymerase trivia

tom chappell tchappell at znet.com
Sun Nov 28 03:07:02 EST 1999


In article <ogzk9MAs9PP4EAGL at genesys.demon.co.uk>, "Dr. Duncan Clark"
<Duncan at genesys.demon.co.uk> wrote:

> In article <tchappell-2411990047050001 at sdts11-124.znet.net>, tom
> chappell <tchappell at znet.com> writes
> >Pfu adds to the ends also, it is just very good at taking it back off
> >again when it realizes it has made a mistake.
> >
> >Whether or not you see it on the end of the DNA product is due to relative
> >rates of pol activity vs. proof-reading activity. 
> 
> Have you a reference on this? I just don't recall any info. from the
> manufacturers and/or suppliers of the 3'-5' exo minus derivatives that
> they add 3' single base overhangs.   
> 
> Has anyone noticed single 3' overhangs when using the exo minus
> derivatives i.e. Vent (exo-) or Deep Vent (exo-)?  By noticed I mean
> proof as in sequencing, not just that they have managed to ligate such a
> product into a TA vector. 
> 
> Duncan

Damn, I hate it when people do this...

I have to look it up and then find that what I remember from DNA
replication (perfectly taught by someone named A. Kornberg, no less...) is
faulty.

T7, Taq, Klenow, Vent and Tth add 3 prime extensions.

Pfu and T4 don't.

Hu, G. (1993). DNA polymerase-catalyzed addition of nontemplated extra
nucleotides to the 3' end of a DNA fragment. DNA Cell Biol 12, 763-70.


However, the exo+ Pfu is 100-fold better than exo- Pfu at preventing
frameshifts in DNA repeats (AAA -> AA or AAA-> AAAA) where the repeats are
NOT at the ends of the fragment. If the repeats are at the end of the
fragment, exo- Pfu can generate 3 prime overhangs, but it does so by first
frameshifting and then reannealing to generate the 3 prime overhang.

Hite, J. M., Eckert, K. A., and Cheng, K. C. (1996). Factors affecting
fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n
microsatellite repeats. Nucleic Acids Res 24, 2429-34.




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