dye labelled primer extension

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Aug 29 03:23:05 EST 2000


In article <8oeche$ng4$1 at pegasus.csx.cam.ac.uk>, Justin Powell
<jacp1 at mole.bio.cam.ac.uk> writes
>Has anyone tried to do 5' transcriptional start mapping using fluorescent
>sequencing technology rather than radioactive?  I am thinking along the
>lines of using dye labelled primers and doing an RT primer extension using
>one of the sequecing primers which would then be run in an adjacent lane
>to the sequencing reaction on an ABI377?
>
>I am wondering whether a) the signal from the primer extension would be
>strong enough, 

Probably actually too strong so you may have to use much less. This is
very similar to when one identifies the cut site for a new restriction
enzyme. You run 5th and 6th lanes next to a sequencing set through the
cut site. The primer extension reactions in lanes 5 and 6 are cleaved
with the RE and then lane 6 is filled (to see any overhang).  The
location of the extension products on the sequencing gel gives the RE
cut site position on both strands. 

>b) whether the adjacent lanes run so that positions
>correspond accurately 

Yes providing you also sequence with dye primers and not dye
terminators.

>and c) whether the analysis software can display the
>two traces side by side or overlayed such that their positions correspond

Pass.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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