Nasty sequencing problem

Joseph C. Bagshaw jbagshaw at wpi.edu
Fri Apr 3 15:34:53 EST 1998


Help!

We are trying to sequence a set of ribosomal RNA genes, and we have just
run into what appears to be a very nasty problem.  We are using the ABI
310 and the Ready Reaction mix, and the problem is in the sequencing
reactions, not the separation.  With some primers we get very good
sequence data for some distance and then the sequence stops abruptly, as
if the polymerase ran into a wall.  I suspect that this problem derives
from the structure of the template, which, in a sequencing reaction,
should take on a briar patch of secondary structure resembling that of the
rRNA.  I think the Taq FS enzyme is not capable of plowing through this
structure.  The problem is clearly related to specific primer sites within
a single plasmid; other primers for IGS sequences in the same plasmid do
not have this problem.

OK, so here's the bottom line: Is there some way we can blast our way
through this briar patch, or are we just rabbit stew?

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.




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