[Cytometry] Flow karyotyping
hms at shapirolab.com
Sun Jul 19 14:12:12 EDT 2009
Derek is correct in that no instrument will be able to discriminate a
derivative chromosome unless its DNA content and/or base composition are
sufficiently different from those of normal chromosomes to place it in a
normally unoccupied spot on a Hoechst/chromomycin bivariate karyotype.
As to whether the lasers on Jeff's Influx would be adequate for
chromosome sorting, Ger van den Engh should be able to provide a
definitive answer. If the 100 mW UV laser is a mode-locked 355 nm, it
should provide enough power because the pulse intensities are
sufficiently high to put the Hoechst dye into photon saturation.
Although the excitation cross section of chromomycin A3 is actually
somewhat higher at 405 nm than at 457 nm, the difference is less than a
factor of two, and 50 mW at 405 nm might not be enough power. Unless the
Vantage is fitted with a mode-locked laser, it probably wouldn't have
enough UV power.
With regard to alternative dyes, I and my colleagues, Tom Frey et al at
BD, and the Los Alamos group all experimented with visible-excited
nucleic acid dyes for bivariate flow karyotyping in the mid-1990s. The
bottom line is that none of the dyes tested then, primarily those from
the YOYO and TOTO series, gave anything like the separation needed for
any serious work. I have, however, recently found in experiments with
bacteria and malaria parasites that, although the combination of an
AT-selective DNA dye (e.g., Hoechst 33342) and a GC-selective DNA dye
(e.g., chromomycin A3) provides the best separation of genomes with
different base compositions, some separation can be obtained using a
Hoechst dye and a visible-excited DNA dye without a base preference,
e.g., PicoGreen. It is probably now appropriate to check some of the
newer DNA-selective dyes, e.g., the Vybrant DyeCycle series, for base
preferences; this is relatively easy to do using the test dye in
combination with a Hoechst dye to stain ethanol-fixed bacteria.
Staphylococcus aureus (69% AT), Escherichia coli (50% AT), and
Pseudomonas aeruginosa (33% AT) provide a test set that spans a
substantially wider range of base composition than is found in human
chromosomes (see pp.317-19 and Figures 7-13 and 7-14 in the 4th Edition
of Practical Flow Cytometry).
Derek Davies wrote:
> Hi Jeff,
> Being able to identify and sort a derivative chromosome will depend on it
> having a different size (DNA content) or bp ratio such that it would move
> from its normal position on a flow karyotype. Small changes, reciprocal
> translocations and so on are often not seen. Traditionally (and we haven't
> sorted a chromosome in anger for quite a while) we used H33258 and
> chromomycin A3. As you say chromomycin is best excited using a 457nm line (a
> rather attractive blue) and I recall using around 300mW of that and UV to
> get best resolution, the more you put in with small stuff the better the
> signal and CV. Havent tried with a violet laser but I suspect the power
> output wouldn't give you the required resolution. I think there have been
> papers using 488nm excited dyes but again I don't think they work that well
> and the Hoechst/chromomycin combination is still the gold standard.
> Best wishes,
> On 16/7/09 08:55, "Jeff Barry" <JBarry at picr.man.ac.uk> wrote:
>> We have a researcher who would like to resolve and sort human
>> isochromosome 17q. From the literature I found that a bivariate plot of
>> Hoechst against Chromomycin A3 seems to resolve chromosome 17 nicely but
>> does anyone know if the i(17q) will form a distinct separate population?
>> I'm also concerned that our present sorters' configurations may not be
>> ideally suited to this, especially regarding the choice of laser lines
>> and power. We have an Influx with 100mw UV and 50wm 407nm laser. I
>> understand that the laser line of choice for Chromomycin is 457nm but
>> has anyone tried with a 407nm line?
>> We also have a Vantage that has a UV laser (<100mw) and an Argon ion
>> laser that could in theory be tuned to 457nm (100wm).
>> Does anyone have an opinion whether these sorters could be used?
>> If anyone has done this before and is willing to share their experience
>> I would be very grateful.
>> Jeff Barry
>> Cancer Research UK
>> Paterson Institute
>> Manchester University
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