Using Pwo DNA polymerase for blunting.

Duncan Clark Duncan at genesys.demon.co.uk
Sat Sep 23 04:50:21 EST 1995


In article: <jpcd0-2209951308540001 at macr1-3.welc.cam.ac.uk>  
jpcd0 at mole.bio.cam.ac.uk (John Dixon) writes:
> 

> 
> Boehringer are also kind enough to include the composition of all the
> components (except the ratio of pwo to Taq) which is nice. Does anyone
> know the optimal ratio? At 56 pounds for thirty eight reactions, we'd 
like
> to make our own.
> 
> No affiliaton etc etc but very impressed by Expand
> 

Pwo is the DNA polymerase from Pyrococcus woesei. I would put money on it 
having >95% homology to Pfu from Pyrococcus furiosus. The bugs were 
isolated by different groups from the same location on the beach at 
Vulcano, Italy. The original papers showed them to be phylogenetically 
pretty close. A more recent paper reckons their DNA:DNA homology is so 
close that they should be the same species. However only the sequence of 
Pfu is in Genbank so time will tell. 

What's the best ratio to use? As you have a working test system buy some 
Pwo and some BCl Taq (sold as different from YTI) and set up seven PCR's 
with 2, 4, 8, 16, 32, 64 and 128 fold dilutions of Pwo to constant Taq. 
Having found which ratio works let us all know!!!

One minor detail, NEB have a patent 53527778 (apart from the VENT one 
5322785) that claims for any  polymerase gene that hybridises to any one 
their VENT gene fragments. If you ever do a DNA alignment of all 
hyperthermophilic DNA pol sequence in Genbank you will find there are very 
few base differences. If you take this to it's logical conclusion Pfu and 
Pwo may infringe that patent. 

Duncan 

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