Overhang A of PCR
jwei at sfu.ca
Tue Apr 29 13:42:09 EST 1997
How about the ULTRA THERM Thermophilic DNA polymerase from Clontech?
> 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
> 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.
> >From various reports mixes of Taq or Tth or KlenTaq and Pfu, or Pwo or
> Vent etc, due to having a low ratio of proof-reading pol to non-proof-
> reading pol activity, will give an A overhang.
> Naming these pols is genrally similar to restriction enzymes. ie
> Thermus aquaticus = Taq
> ^ ^^
> Thermus thermophilus = Tth
> ^ ^^
> Pyrococcus furiosus = Pfu
> ^ ^^
> Vent and Deep Vent are trade names. Vent is from Thermococcus litoralis
> and Deep Vent from a Pyrococcus species.
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
> Duncan Clark
> DNAmp Ltd.
> TEl/FAX 01252376288
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