compressions during sequencing

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Jun 30 10:33:42 EST 1994


>On 24 Jun 1994 13:55:05 -0700,
>  Darrick Carter writes:
>
>
>
>Does anyone have a good suggestion about how to sequence a very G-C rich
>genome? We've tried formamide in the gel and deaza-guanine, but even
>these don't seem to get rid of the compressions. Any help?
>
Sometimes it's not so much compression as it is the polymerase having
trouble getting through the G+C regions in the template.  (ie. gel
spacing is all right, just bands in all lanes in G+C runs). Sometimes
it's hard to tell these apart (and sometimes you have both at once).
A true compression (disruption of spacing while the fragment runs
on the gel because it hairpins) tends to be given away by a decompression
(bands too far apart) just above it on the gel. 
In the case where polymerase stoppage is contributing,
we've had success with cycle sequencing, presumably because the
high temperature melts out the template structure.

I'd appreciate it if you post a summary of anything that you find that
actually works.  We fight this problem a lot, and frankly often end up
relying on the sequence of the other strand.  For a true compression,
it is helpful to realize that the problem will
set in as you hit the 2nd component of the hairpin.  So the compression
on the other strand will often be displaced.

                                   Compressed on strand 1
                                      |
               ----------GGGGGG----CCCCCC-------->
              <----------CCCCCC----GGGGGG---------
                          |
                     Compressed on strand 2

  So sometimes, but not always, you can figure out which strand to believe
at all positions.

Good luck.
Steve Hardies    Hardies at thorin.uthscsa.edu




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