Re: chromosome sorting

From: Howard Shapiro (hms@shapirolab.com)
Date: Wed May 31 2000 - 20:44:06 EST


Julie Auger asks:


>Has anyone tried sorting chromosomes using Acridine Orange and 7-AAD?  We
>want to sort Indian muntjac chromosomes (only 3  or 5 chromosomes per
>cell).  Any advice would be appreciated.

Why AO and 7-AAD? If the idea is to get a 2-color bivariate histogram, AO
probably wouldn't be the best choice because it has high background
fluorescence, is metachromatic, and - if I remember correctly - doesn't
have much base pair specificity.  I've tried 7-AAD on chromosomes, and the
staining is very weak, and not, at least in my hands, particularly
base-pair specific.  Both Tom Frey at B-D and I got some bivariate staining
of human chromosomes with combinations of 488-excited and red-excited
Molecular Probes dyes (TOTO-1 and TOTO-3 in my case, thiazole orange and
thiazole blue in Tom's), and that might be worth a shot.  These dyes aren't
DNA-specific, but chromosomes don't have appreciable RNA.  But maybe the
whole bivariate thing is overkill; I seem to recall that Muntjac
chromosomes can be well separated using a single stain, such as PI.  It
might be that a single Molecular Probes dye, such as Pico Green, would be
equally useful.

-Howard



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