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>I would like to add anoather question concerning chromosome sorting. Is
>there a second dye that can be paired with propidium iodine for two color
>and be excited with a 488 laser? Or two other dyes that bind DNA and can
>be excited at 488?
The short answer is no. The problem is finding a visible-excited dye which
has the A-T preference of the Hoechst dyes and DAPI and another which has
the G-C preference of the chromomycins (A3, mithramycin, olivomycin).
Without base preferences, you'd get a bivariate histogram which would put
all the chromosome clusters along a 45 degree line (assuming equal gains for
both fluorescence signals); the base preference and the different A-T/G-C
ratios of similarly sized chromosomes, which moves them off the line, are
what makes bivariate analysis and sorting of chromosomes work.
The Los Alamos group managed to get movement off the line using Hoechst and
YOYO-1 or TOTO-1; they also got some movement with the YOYO or TOTO dyes
alone, but that probably involved selective bleaching in the first beam, as
they use very high laser powers.
Tom Frey et al at B-D looked at thiazole orange, thiazole blue, and the TOTO
dyes, although their publication was mostly oriented toward use of He-Cd
lasers for UV excitation (Frey T, Houck DW, Shenker BJ, Hoffman RA:
Bivariate flow karyotyping with air-cooled lasers. Cytometry 16:169-74, 1994).
We've been looking for a visible-excited dye combination in my lab for
several years; at one point, TOTO-1 (488 excitation, A-T preference) and
TOTO-3 (633 excitation, G-C preference) looked promising, but we tried it on
an Elite and didn't get much separation in the bivariate histogram. We have
found a few other cyanine and styryl dyes with base preferences, but neither
we nor Tom Frey (with whom we shared the information) have obtained anything
useful in the way of bivariate chromosome histograms. We're still looking.
Dick Haugland et al at Molecular Probes have been very helpful in letting us
test their new nucleic acid stains; the laser dye manufacturers have also
donated dyes. The bottom line is that appropriate dyes, if they exist, will
probably have to be designed based on spectral information now available.
The effort would probably also yield a "visible excited Hoechst 33342),
i.e., a dye which would be DNA-specific enough to give a passable DNA
histogram in viable cells without fixation or permeabilization. My guess is
that if Dick though it would be reasonably easy to do he would have
submitted an SBIR application on the subject; as far as I know, that hasn't
happened yet, but hope springs eternal.
-Howard
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