Pfu-spiked cycle sequencing

[Duncan Clark]

In article: <9406091406.AA03996 at deimos.med.pitt.edu>  rapr at MED.PITT.EDU (Robert Preston) writes:
> 
> Forwarded message:
> > From: ohungnes at bioslave.uio.no (Olav Hungnes)
> > 
> > Robert Preston (rapr at MED.PITT.EDU) wrote:
> > : Forwarded message:
> > : > From: jspaffor at gpu.srv.ualberta.ca (J. David Spafford)
> > : > [edit] 
> > : > I use an ABI sequencer with a 9600 Perkin Elmers PCR machine.  Usually I can
> > : > get up to 400 bp of good sequence and no more.  ABI provides AmpliTaq with
> > : > their sequencing kit.  Would I get longer sequence if I spike it with Pfu
> > : > DNA polymerase?
> > : > J. David Spafford
> > 
> > : You mean, do Wayne Barnesian Long-PCR factors come into play in the 400-700
> > : bp range?  Interesting possibility.  Well worth an experiment.  Be sure to
> > : post the results!
> > : Rob Preston
> >
> > I don't know much about Pfu, but in the good old days of Vent I was told 
> > that the 5'to 3' proofreading exonuclease activity would eat at all kinds of 
> > non-base-paired 3' termini, even the primers to some extent. Isn't there 
> > a chance that the Pfu will start degrading dideoxy-terminated molecules 
> > from their 3' end and make a mess of the entire reaction?
> > Olav
> > --
> There seems to be some confusion about 5' and 3' there, Olav, but more
> to the point, why would dideoxyterminated strands be degraded? They are
> not mis-paired (we use them for sequencing, after all), so presumably they
> would not be editor substrates.  They just can't be extended further.  In
> any case, even if they were edited sometimes, further synthesis on the
> same strand would likely fix things up (for fluor primer chemistry) or it
> would be irrelevant (for fluor terminator chemistry).  Even MORE to the point,
> one experiment (well, maybe two or three tries, if it's as simple as these)
> is worth far more than any of this verbal rationalization..
> Rob Preston
> rapr at med.pitt.edyu
> 
Although this is going to be tried I still think that a proof-reading pol 
with its 3'-5' proofreading exonuclease (not 5-3) will remove the dideoxy
terminators. This is the reason why both NEB and Stratagene produce an exo-
version of VENT and Pfu for sequencing. The other reason is that the proof
reading exo. can shorten primers from the 3' end. DideoxyCTP inhibition has
been used for a long time to distinguish different DNA pols and also their
proofreading capabilities. Obviously the experiment will sort all this out,
just remember to use only trace amounts of the Pfu.

Good luck.

Duncan 
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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
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