GC rich sequencing

pdxrmb at vaxb.ccc.nottingham.ac.uk pdxrmb at vaxb.ccc.nottingham.ac.uk
Fri Jul 25 09:43:02 EST 1997


I have been sequencing high GC PCR products (70-85%) for a few months and have gone back to manual cycle sequencing, because the elimination of stops and com
pressions is only complete when using 7-deaza GTP in cycle sequencing react-
ions (in my experience). If you can get clean PCR of your insert, uisng high
annealing (60-70 celsius) primers, these can be used to sequence the insert
with the same cycle parameters as the amplification. We are using the USB
Thermosequenase cycle sequencing kit, with P33 end labelled primers and i
haven't seen a compressio/secondary structure induced stop for months. Also
we had a trial of the new P33 labelled dideoxy terminators (also USB/Amer-
aham) with the thermosequenase cysle kit. This was by far the best we've 
tried- the sequence starts at the base after the primer, the deaza (or sITP
mixes) sort out gel compressions, and the fact that only dideoxy terminated
extension products are labelled means no stops! If you are going to want-
ing to do a lot of high GC sequencing this seems to be a good system. If its
only A few templates (and you don't want to go hot) than maybe the Thermo-
sequenase dye terminator kits would be best.
Hope this helps- 

Richard M. Badge 



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