Overhang A of PCR

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Apr 29 07:49:43 EST 1997


In article <3364FDAE.1BD7 at mindspring.com>, "Matthew L. Brown, Ph.D"
<gemini3 at mindspring.com> writes
> I
>believe (and someone can correct me if I'm wrong because I don't feel
>like looking it up) that Klenow, Pfu, and Pwo all have 3' to 5'
>exonuclease activity.

For Klenow I assume you mean KlenTaq.

Quick overview of enzymes used in PCR:

Vent, Pfu, Pwo, Tli and Deep Vent are hyperthermophilic archaebacterial
DNA polymerases. All possess a 3'-5' proof-reading exonuclease activity.
Because of this they offer lower errors and will not add the extra A
overhang.

The exo-variants of the above are deletions or mutations of the 3'-5'
exonuclease domain. I'm not sure whether they give an A overhang. The
archael enzymes are totally different beasts to the Thermus and
Thermotoga pols.

Taq, Tfl, Tth are all thermophilic bacterial DNA polymerases isoloated
from a varity of Thermus strains. They do not possess a 3'-5'
exonuclease and therefore do not proof-read but they do have a 5'-3'
exonuclease activity (as used in the TaqMan system). They all have the
associated terminal transferase activity and add preferentially a single
A (*) overhang. 

Klentaq and Stoffel are 5'-3'exonuclease minus deletions of Taq losing
the first 260-290 aa at the NH2 end which is where the exo. domain is.
If you go to the PDB you can get Xtal structures for Taq and Stoffel and
the exo and pol domains are clearly seen.

UlTma is a hyperthermophilic bacterial enzyme isolated from Thermotoga
maritima. It has a proof-reading 3'-5' exonuclease but from all the
published fidelity results I have seen in NAR or in Strategene adverts,
seems to have less fidelity than Taq! Due to the exo. activity I don't
think it will give the A overhang.



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