Pfu-spiked cycle sequencing

Olav Hungnes ohungnes at bioslave.uio.no
Tue Jun 14 04:41:42 EST 1994


Robert Preston (rapr at MED.PITT.EDU) wrote:
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

Yes, I got the 5' and 3' ends wrong, sorry. Still, isn't this cycle 
sequencing? The 3' ends may be 
available for degradation, not because they are mismatched (they are 
not), but because they are single-stranded; late in the reaction there 
will be an excess of extended primer strands over template strand. This 
will also make further synthesis of the partially degraded strand less likely.
But, I would also like to see the experiment done, just watch the Pfu 
concentration. 

-- 
_______________________________________________________
Olav Hungnes                     ohungnes at embnet.uio.no
National Institute               Phone  (+47)22042200
of Public Health                 FAX    (+47)22353605
Oslo, NORWAY
_______________________________________________________



More information about the Methods mailing list