Sequencing high G + C DNA

William Bonner wmbonner at helix.nih.gov
Tue May 9 19:15:44 EST 1995


When we had problems with a high GC region, what finally worked for us
after trying alot of things was to use single-strand binding protein
in the rx during elongation.  I think it's mentioned in the Sequenase
booklet in the back.  Good luck-Bill Bonner

Steven Goldberg <goldberg at bms.com> wrote:

>I have been attempting to sequence DNA of high G + C content (ca. 72%)
>that has been cloned into various E. coli plasmids.  My main problems
>(couldn't you guess?) have been weak signals and stops as well as
>phantom bands across all of the lanes in some instances.
>The manner in which the DNA has been prepared doesn't seem to make
>much difference, as I get the same results using CsCl-purified, Qiagen-
>purified, or mini-preped plasmid.  I started off using the basic 
>protocol given with the Sequenace kit + deazaGTP and 35S-dCTP.  I 
>have also tried the Quick Denature kit with the same parameters.  
>Primers to plasmid or cloned DNA were made but didn't make much 
>difference.  Someone suggested including 1 ul DMSO in my reactions,
>which did seem to help the stops a little (but could it also knock
>down the activity of the polymerase?).  I raised the termination
>reaction temperature to 42oC.  All of these changes really didn't
>help a lot.  How about using 32P instead 35S?   

>If anyone with a similar experience has any ideas, I and our radiation
>safety department would be grateful.  Thanks.

>Steve Goldberg 





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