Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/

Pairs of fluorescent dyes as probes of DNA and chromosomes. S A Latt, E Sahar and M E Eisenhard J Histochem Cytochem 1979 27: 65 DOI: 10.1177/27.1.86582 The online version of this article can be found at: http://jhc.sagepub.com/content/27/1/65

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0022-

1554/79/2701

-0065/$02.OO/()

THE JOURNAL OF HISTOcHEMISTRY Copyright © 1979 by The Histochemical

Pairs

AND

Inc.

of Fluorescent SAMUEL

Center

Vol. 27, No.

CYTOcHEMISTRY

Society,

for Mental

Retardation

Dyes A. LATT,

and

as Probes

ELHANAN

Department

SAHAR

ofMedicine, Received

of DNA MICHAEL

AND

Children’s

Hospital

for publication

and

June

1, pp. 65-71, 1979 Printed in (ISA.

Chromosomes1

E. EISENHARD Medical

Center,

Boston,

Massachusetts

02115

5, 1978

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions. stain

A large with and are

number

of fluorescent

fluorescence DNA present which DNA

capable

of interacting

can be influenced not only by but by DNA base composition, is substituted by base analogues,

restriction

of

However,

DNA

accessibility

if two

same object, as a normalized

the

dyes ratio measure

Normalization

of a dye content,

tion-insensitive measure Especially when used analysis, dual fluorochrome resolution

of flow

30, 36). For many

dye

pairs,

inter-dye

dye pairs for either

chromosomal

paper

to

can

in principle

of the total in conjunction staining studies

sufficient

MATERIALS

the

can serve features. be achieved

iO

property, such as a composi-

electronic

1, 5, 9, 1 1, 20, excitation

nm)

dures,

28,

However,

energy

meaproor

transfer

of proximity relationships about distances between

or about clustering of sites in chromatin energy donor or acceptor dyes. The present

I Supported by Grant GM21121 from The National Health. SAL. is the recipient of a National Institutes Research Career Development Award GM 00122.

from

AND

information dyes

used

about in

pairs,

due to electronic to assess intensive

METhODS

(ethidium, (7),

480

nm)

(2),

21.5

x

iO

(7-aminoactinomycin

D,

502

8.85

with

or without

added

bromodeoxyuridine

(BrdU),

slides

were

prepared by air drying, and the fluorescence of stained chromosomes was observed with a Leitz Orthoplan microscope equipped with incident illumination (11). Absorption and fluorescence spectra were obtained with Cary Model 118C and Hitachi MPF-3 spectrometers, respectively. Energy transfer measurements on stained cytologic metaphase chromosome preparations utilized a Leitz MPV II microspectrofluorometer. Samples were exposed to incident illumination from a 200 watt mercury lamp, using a X 25 objective, and the fluorescence intensity from a single entire metaphase, delimited by a square aperture, was measured within 10 eec, to minimize fading (