primer extensions by fluorescence

Michael Kertesz kertesz at micro.biol.ethz.ch
Fri Jun 20 10:31:06 EST 1997

I have been wondering if anyone has done RNA primer extension analyses
using fluorescent dye-labelled primers for the RT reaction, followed by
analysis on e.g. an ABI sequencer. It would seem an easy way to do it
non-radioactively, and the size of the resulting cDNA could be calibrated
by spiking it into a normal sequencing run made using the same primer,
hence determining the transcription start site. To get round sensitivity
problems, it might be possible to incorporate dye-labelled NTP's, rather
than using dye-primers, hence increasing the fluorescence yield. BUT.... if
it were that easy, everyone would be doing it, right?. Has anybody tried
this out, and how were the results?

thanks a lot in advance



Michael Kertesz

ETH Institute of Microbiology,	tel: +41-1-632 3357
ETH-Zentrum/LFV,				fax: +41-1-632 1148
CH-8092 Zurich, Switzerland		e-mail: kertesz at micro.biol.ethz.ch

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