Summary
The dye triplet chromomycin/methyl green/DAPI and some related dye combinations were applied to bromodeoxyuridine (BrdU)-substituted and non-substituted chromosomes. In both kinds of chromosome preparations, tristaining followed by observation at pH 7 resulted in well defined R-bands (excitation wavelength 436 nm) and a mixed DA-DAPI/Q-banding pattern (360 nm). Two approaches have given satisfactory expression both of reverse bands and of the differential BrdU-substitution present in metaphase chromosomes: (1) direct tri-staining at pH 7 and mounting of the preparations at pH 11; or (2) pretreatment of the preparations with Hoechst 33258 plus UV and hot buffer followed by tri-staining and mounting at pH 7. These methods should prove useful for routine chromosome analysis and, in combination with BrdU-labelling, in studies of chromosome structure and replication.
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References
Ambros P (1983) Untersuchungen zur Feinstruktur mitotischer Chromosomen: Replikationsmuster, Verteilung der Schwesterchromatid-Austausche und Bänderungsverhalten bei Ornithogalum (Liliaceae). PhD Thesis, University of Vienna
Buys CHCM, van der Veen AY (1982) Different effects of 33258 Hoechst and DAPI in fluorescence staining of sister chromatids differentially substituted with bromodeoxyuridine. Histochemistry 75:169–177
Calderon D, Schnedl W (1973) A comparison between quinacrine fluorescence banding and 3H-thymidine incorporation patterns in human chromosomes. Hum Genet 18:63–70
Crossen PE (1983) DNA replication and sister chromatid exchange in 9qh+. Cytogenet Cell Genet 35:152–155
Crossen PE, Drets ME, Arrighi FE, Johnston DA (1977) Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes. Hum Genet 35:345–352
Donlon TA, Magenis RE (1983) Methyl green is a substitute for distamycin A in the formation of distamycin A/DAPI C-bands. Hum Genet 65:144–146
Dutrillaux B, Fosse AM (1974) Sur le mechanisme de la segmentation chromosomique induite par le BUDR (5-bromodeoxyuridine). Ann Genet (Paris) 17:207–211
Dutrillaux B, Viegas-Pequignot E (1981) High-resolution R- and G-banding on the same preparation. Hum Genet 57:93–95
Haglund U, Zech L (1979) Simultaneous staining of sister chromatid exchanges and Q-bands in human chromosomes after treatment with methyl methane sulfonate, quinacrine mustard and quinacrine. Hum Genet 49:307–317
Hoo J, Parslow PI, Chambers D (1979) Combination of differential sister chromatid staining, G-banding pattern, and X-inactivation pattern. Hum Genet 47:291–295
Kato H (1974) Spontaneous sister chromatid exchanges detected by a BUdR-labelling method. Nature 251:70–72
Kroisel PM, Rosenkranz W (1984) Simultane Fluoreszenzfärbung von Schwesterchromatidaustauschen (SCE) und R- bzw. Q-Bändern an menschlichen Chromosomen. (Abstract) 9. Tagung Sekt. Cytogen. Ges. Anthropologie u. Humangenetik, GFR, Mainz
Kurnick NB, Foster M (1980) Methyl green. III. Reaction with deoxyribonucleic acid, stoichiometry, and behavior of the reaction product. J Gen Physiol 34:147–159
Latt SA (1973) Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395–3399
Lin MS, Alfi OS (1976) Detection of sister chromatid exchanges by 4′-6-diamidino-2-phenylindole fluorescence. chromosoma 57:219–225
Lin MS, Comings DE, Alfi OS (1977) Optical studies of the interaction of 4′-6-diamidono-2-phenylidole with DNA and metaphase chromosomes. Chromosoma 60:15–25
Müller W, Gautier F (1975) Interactions of heterochromatic compounds with nuclei acids. A-T-specific non-intercalating DNA ligands. Eur J Biochem 54:385–394
Ockey CH (1980) Autoradiographic evidence of differential loss of BUdR-substituted DNA after UV exposure in FPG harlequin staining. Exp Cell Res 125:511–514
Pathak S, Stock AD, Lusby A (1975) A combination of sister chromatid differential staining and Giemsa banding. Experientia 31:916–918
Perry P, Wolff S (1974) New Giemsa method for the differential staining of sister chromatids. Nature 251:156–158
Sahar E, Latt SA (1978) Enhancement of banding patterns in human metaphase chromosomes by energy transfer. Proc Natl Acad Sci USA 75:5650–5654
Sahar E, Latt SA (1980) Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes. Chromosoma 79: 1–28
Schempp W (1980) Asynchrony in late replication between homologous autosomes in primary cultures of Chinese hamster fibroblasts. Chromosoma 79:199–206
Schempp W, Vogel W (1978) Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU. Chromosoma 67:193–199
Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58:307–324
Schweizer D (1980) Simultaneous fluorescent staining of R bands and specific heterochromatic regions (DA-DAPI bands) in human chromosomes. Cytogenet Cell Genet 27:190–193
Schweizer D (1981) Counterstain-enhanced chromosome banding. Hum Genet 57:1–14
Schweizer D (1983) Distamycin-DAPI bands: properties and occurrence in species. In: Brandham PE, Bennett MD (eds) Kew Chromosome Conference II. Georg Allen and Unwin. London, pp 43–51
Webber LM, Brasch JM, Smyth DR (1981) DNA extraction during Giemsa differentiation of chromatids singly and doubly substituted with BrdU. Chromosoma 81:691–700
Zakharov AF, Egolina NA (1972) Differential spiralisation along mammalian mitotic chromosomes. I. BUdR-revelaed differentiation in Chinese hamster chromosomes. Chromosoma 38:341–365
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Kroisel, P.M., Rosenkranz, W. & Schweizer, D. Simultaneous production of R-bands and either replication patterns or sister chromatid differentiation. Hum Genet 71, 333–341 (1985). https://doi.org/10.1007/BF00388460
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DOI: https://doi.org/10.1007/BF00388460