We have run into problems sequencing a cDNA clone from
Chlamydomonas eugametos (which has a GC content of about 60-65%).
Almost everything is sequenced except for a 100-150 bp fragment
(according to the restriction map). The T7 polymerase sequence
reaction gives rise to an aspecific band in every lane. We tried to
solve the problem (on both strands) by using Taq polymerase and 72
degrees celcius, deaza-sequencing and making primers just beside
the block. Nothing worked.
We are pretty sure that the above problem is caused by the strong
secundairy structure because:
- Control DNA isolated at the same time with the same
protocol worked just fine.
- The deaza and Taq sequencing didn't solve the problem.
- The extremely low abundancy of the clone in the cDNA
library (1 in every 1.5 million independent clones).
Probably the RT reaction also encountered the strong
Currently we are trying to make Exo III deletion clones by removing
a few bases at a time. We don't yet know if the Exo III deletions
will solve the problem, but before that also fails, we would like
to have an alternative.
- So are there other people who have the same problem?
- What was the solution?
- Is chemical sequencing an alternative? If so, who has a
working protocol? Is it easy to set up in a lab with no
prior experience with this protocol?
Any help is appreciated. If there is enough interest in this suject
I will post an abstract if desired.
Henk van de Kamer,
University of Amsterdam.
CHLAMY at SARA.NL