Nasty sequencing problem

[Alexander Kraev]

In article <Pine.OSF.3.96.980403152333.21572A-100000 at reno.WPI.EDU>,
"Joseph C. Bagshaw" <jbagshaw at wpi.edu> wrote:

> 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?

Joe, there are two known solutions to this problem:

1. Add 5-10% DMSO to the reaction

2. Treat the template with Thermofidelase (http://www.fidelitysystems.com/)

which improve the results dramatically but still not to 100%

good luck,

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch