High temp sequencing

Brain Foley brianf at med.uvm.edu
Fri Aug 6 10:16:55 EST 1993


GRGGTA at PATERSON.CHRISTIE-HOSPITAL.MANCHESTER.AC.UK wrote:
: Jon Jafari writes

: Sorry if this is a FAQ, but I couldn't find a FAQ for this group.  

The FAQ can be obtained from fly.bio.indiana.edu via anonymous FTP or via
GOPHER.  I will mail you a copy of it.

: I am having serious proplems with secondary structure and would like
: to know if anyone has used taq to label at a higher temp (or
: sequenase) with success and if so what were the conditions?  I have
: called Promega re their taq kit but they "dogged me like toto" and
: sent the same information that was in their catalog.  Thanks for your
: help.

I have used sequenase at temps up to 45 degrees centigrade with much
success.  The higher temp does help get through secondary structure.  The
use of 7-deaza-GTP inplace of dGTP in the reActions also made significant
improvements.

I have also used some thermostable polymerases (TAQ, Tth and Vent) for
sequencing at temps near 75 degrees centigrade.  These polymerases got
through the secondary structures just fine, but they seemed to give less
even band intensities that sequenase.  One advantage of the thermostable
polymerases is the technique of "cycle sequencing" where you elongate
once, then heat to 95 degrees, cool to anneal again, elongate again, etc...
Cycle sequencing has been extensively described in the literature, so I
won't post my protocol here.  The major advantage of cycle sequencing is
the ability to sequence from a much smaller amount of template DNA.  The
disadvantage is that it is not a "tried and true and foolproof" as the
standard methods using sequenase.  The ratios of dNTPs to ddNTPs given in
various protocols for thermostable polymerases vary greatly.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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