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

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


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

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