Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP)

wegloehner at mpimg-berlin-dahlem.mpg.de wegloehner at mpimg-berlin-dahlem.mpg.de
Tue Sep 3 03:25:34 EST 1996


In article <50fjm3$db1 at sparcserver.lrz-muenchen.de>, diabetes at lrz.uni-muenchen.de (Wolfgang Schechinger) writes:
> Hi folks! Sorry for the long subject.
> 
> Actually I wanted to use a kit from Amersham with 33P radiolabelled
> ddNTPs to overcome my sequencing problem: unspecific terminations
> -staggering enzyme- in a GC rich sequence. Unfortunately my lab
> doesn't have the permit to use 33P yet. Thus I'm looking for hints on
> other kits or methods to overcome this problem. Yet I have tried 
> * cycle sequencing from plasmid DNA with 7-deaza GTP
> * "convential" plasmid sequencing (dITP, dGTP)
> * lowering the termination temperature from 37C to 30C to prevent the
> polymerase from slipping off the DNA
> * running a 40% formamide gel
> 
> but always I get these unspecific terminations.
> 
> Now I want to try another way of radiolabelling the sequencing
> products by using radioactive ddNTPs in order to see only the
> correctly terminated DNA transcripts on the gel. But not using 33P.
> 32P or 35S is OK. (Maybe we're kind of stoneage here?)
> Who can help or give hints? YOUR opinion is very appreciated!
> 
> please mail and post your reply.
> 
> Wolfgang Schechinger
> Institute for Diabetes Research
> Kölner Platz 1
> 80804 Muenchen
> Germany
> Phone +49 (89) 30 79 31 24
> Fax            30 81 73 3
> E-mail: DIABETES at LRZ.UNI-MUENCHEN.DE
> 

Why don't you try cycle sequencing on an automated sequencer using labelled
ddNTPs. And if there isn't a ABI machine in your institute phone Applied
Biosystems, they should be able to give you a hint were in muenchen you can
find one. 
We had exactly the same problem - GC contents of more then 90% in a stretch of
over 200 bp. The only way to get the sequence was cycle sequencing with
labelled ddNTP. At least in our hands this worked fine - even without any
experience - we used a borrowed kit and gave the samples to the technician who
was running the machine.
hope this helps

wolf








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