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Fluorescence R-Banding

R Banding by Fluorescence Using Acridine Orange (AO)


R-banding methods are useful for analyzing deletions or translocations that involve the telomeres of chromosomes.


Acridine orange was originally used to stain untreated chromosomes, both human and mouse. Bobrow et al. and Baserga and Castoldi independently reported the use of acridine orange to obtain a reverse banding pattern of chromosomes. Acridine orange (AO) is a base composition-independent fluorochrome that binds to DNA by intercalation and which gives relatively uniform fluorescence along the length of the chromosome arms. The dye binds very little to non-nucleic acid cell components, but it fluoresces orange-red when bound to single-stranded nucleic acids and yellow-green when bound to double-stranded nucleic acids. Following hot phosphate buffer treatment, R bands are yellow-green, and G/Q bands are orange-red. The major factor that contributes to R banding is the relative GC-richness of the R bands. In many laboratories, RHG methods have been abandoned in favor of a fluorescent R-banding technique (Gustashaw, 1991).


  • Phosphate Buffer:
    32 ml of 0.07N Na2HPO4 . 12 H20
    68 mL of 0.07 mol/L KH2PO4
    Adjust pH to 6.5 by adding 0.07N Na2HPO4 . 12 H2O to the solution.
  • Acridine Orange: 0.01%, prepared in the phosphate buffer


  1. Prewarm the buffer to 85 degrees C.

  2. Incubate one slide for 10 to 30 minutes in hot phosphate buffer.

  3. Stain with 0.01% acridine orange for 4 to 6 minutes.

  4. Rinse in phosphate buffer (pH 6.5) for 1.5 to 3 minutes.

  5. Mount with the some buffer, without sealing the coverslip.

  6. Examine by fluorescence microscopy using the appropriate filter combination (e.g., excitation: 450-490 nm; suppression: 515 nm).


Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual, Second Edition, edited by M. J. Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.

Verma, RS, Lubs HA. Additional observations on the preparation of R banded human chromosomes with acridine orange. Can J Genet Cytol 18:45-50, 1976.

Verma, RS, Lubs HA. A simple R banding technique. Am J Hum Genet 27:110-117, 1975.

Primate Cytogenetics Network