dye labelled primer extension

Arnoud van Vliet avvliet at knoware.nl
Mon Aug 28 15:25:10 EST 2000


> 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?

Dear Justin

we have been thinking about this as well. I have seen a reference where they
used fluorescent primers and an ALF sequencer for primer extension. The
references are:

Grass, G., C. Große, and D. Nies. 2000. Regulation of the cnr cobalt and
nickel resistance determinant of Ralstonia sp. strain CH34. J. Bacteriol.
182:1390-1398.

Große, C., G. Grass, A. Anton, S. Franke, A. Navarrete Santos, B. Lawley, N.
L. Brown, and D. H. Nies. 1999. Transcriptional organization of the czc
heavy metal homoeostasis determinant from Alcaligenes eutrophus. J.
Bacteriol. 181:2385-2393

If you get the primer extension running, we'd be very interested in the
protocol, and maybe you can post it on the newsgroup?

Hope this helps
Arnoud







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