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Chromosome Banding Techniques

Rhona R. Schreck¹, Christine M. Distèche²

¹Cedars‐Sinai Medical Center, Los Angeles, California
²University of Washington School of Medicine, Seattle, Washington

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 4.2

DOI:

10.1002/0471142905.hg0402s00

Online Posting Date:

May, 2001

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Abstract

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.

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Unit Introduction
Strategic Planning
Basic Protocol: Quinacrine Banding (Q-Banding)
Reagents and Solutions for Q-Banding
Commentary for Q-banding
Giemsa Banding (G-Banding)
Basic Protocol: GTG Technique for G-Banding
Alternate Protocol 1: G-Banding with Wright Stain
Support Protocol 1: Aging Slides with Heat
Support Protocol 2: Aging of Slides with Hydrogen Peroxide
Reagents and Solutions for G-Banding
Commentary for G-Banding
Reverse Banding(R-Banding)
Basic Protocol: RHG Technique for R-Banding
Alternate Protocol 2: CA3/DA Technique for R-Banding
Reagents and Solutions for R-Banding
Commentary for R-Banding
Replication Banding
Basic Protocol: B-Pulse Replication Banding for Lymphocytes
Alternate Protocol 3: T-Pulse Replication Banding for Lymphocytes
Alternate Protocol 4: B-Pulse Replication Banding for Fibroblasts
Alternate Protocol 5: T-Pulse Replication Banding for Fibroblasts
Support Protocol 3: Visualization of BrdU Replication Banding by Fluorescent Dye
Support Protocol 4: Visualization of BrdU Replication Banding by Light Treatment and Giemsa Staining
Support Protocol 5: Visualization of BrdU Replication Banding by Heat Treatment and Giemsa Staining
Reagents and Solutions for Replication Banding
Commentary for Replication Banding
Basic Protocol: Centromeric Heterochromatin Staining (C-Banding)
Reagents and Solutions for C-Banding
Commentary for C-Banding
Basic Protocol: Nucleolar-Organizer-Region Staining (NOR Staining)
Reagents and Solutions for NOR Staining
Commentary for NOR Staining
Distamycin-DAPI Staining
Basic Protocol: Distamycin-DAPI Staining
Alternate Protocol 6: Hoechst 33258–Distamycin Staining
Reagents and Solutions for Distamycin/DAPI Staining
Commentary for Distamycin/DAPI Staining
Sequential Staining of Slides
Basic Protocol: Sequential Staining
Support Protocol 6: Destaining
Commentary for Destaining
Bibliography
Figures
Tables

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Materials

Basic Protocol: Quinacrine Banding (Q-Banding)

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Air-dried slides of metaphase chromosomes (unit 4.1)
Quinacrine staining solution (see recipe)
McIlvaine buffer, pH 5.6 (see recipe)
Immersion oil, low fluorescence
Coverslips, no. 0 or no. 1

CAUTION: Quinacrine is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol: GTG Technique for G-Banding

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

HBSS (appendix 2D)
Trypsin solution (see recipe)
70% and 90% (v/v) ethanol
2% Giemsa staining solution (see recipe; prepare fresh)
Aged slides of metaphase chromosomes (see support protocols)
Xylene or Hemo-De

CAUTION: Xylene is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

NOTE: Because the stain is difficult to remove from skin, it is advisable to wear gloves when working with Giemsa.

Alternate Protocol 1: G-Banding with Wright Stain

Additional Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Bacto Trypsin solution (see recipe)
Sorensen phosphate buffer, pH 6.8 (see recipe)
Wright stain (see recipe; prepare fresh)

Support Protocol 2: Aging of Slides with Hydrogen Peroxide

Materials

Air-dried slides of metaphase chromosomes (prepare freshly; unit 4.1)
15% (v/v) H₂O₂ (dilute 30% H₂O₂ 1:1 with water immediately before use)
50°C hot plate or slide warmer

CAUTION: H₂O₂ is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol: RHG Technique for R-Banding

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

1.25 M sodium phosphate buffer, pH 4.0 (see recipe)
Air-dried slides of metaphase chromosomes (prepare fresh; unit 4.1)
10% Giemsa staining solution (see recipe)
Coplin jars
88°C circulating water bath with cover

Alternate Protocol 2: CA3/DA Technique for R-Banding

Additional Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Chromomycin A3 staining solution (see recipe)
Distamycin A staining solution with MgCl₂ (see recipe)
Glycerol
Coverslips, no. 1

Additional reagents and equipment for fluorescence microscopy (unit 4.4)

Basic Protocol: B-Pulse Replication Banding for Lymphocytes

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Phytohemagglutinin (PHA)-treated lymphocyte cultures (unit 4.2)
10 mM BrdU (30.7 mg in 10 ml H₂O; store in 1-ml aliquots £6 months at –20°C)
10 mM 2¢-deoxycytidine (dC; 28 mg in 10 ml H₂O; store in 1-ml aliquots £6 months at –20°C)
10 µg/ml Colcemid (e.g., GIBCO/BRL; store at 4°C)
Tissue culture vessel of desired size

Additional reagents and equipment for culture and metaphase harvest of peripheral blood (unit 4.1) and chromosome slide preparation (unit 4.1)

CAUTION: Human lymphocytes and BrdU are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Alternate Protocol 3: T-Pulse Replication Banding for Lymphocytes

Additional Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Tissue culture medium with and without 10 µM thymidine (from 1 mM thymidine stock; unit 8.3), 37°C
IEC HS-NII centrifuge with 958 rotor (or equivalent)

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Alternate Protocol 5: T-Pulse Replication Banding for Fibroblasts

Additional Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Actively growing fibroblast cultures (~8 to 10 hr after passage)
Tissue culture medium with and without 10 µM thymidine (from 1 mM thymidine stock; unit 8.3), 37°C

CAUTION: Human fibroblasts are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 3: Visualization of BrdU Replication Banding by Fluorescent Dye

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Metaphase slides of BrdU-substituted chromosomes (preceding basic or alternate protocols)
95%, 70%, and 30% (v/v) ethanol
PBS (appendix 2D)
Hoechst 33258 staining solution A (see recipe)
McIlvaine buffer, pH 7.5 (see recipe)

CAUTION: Hoechst 33258 is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 4: Visualization of BrdU Replication Banding by Light Treatment and Giemsa Staining

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Metaphase slides of BrdU-substituted chromosomes (preceding basic or alternate protocols)
PBS (appendix 2D)
Hoechst 33258 staining solutions A and B (see recipe)
2× SSC (appendix 2D), 60° or 65°C
4% Giemsa staining solution (see recipe)

100 × 100 × 15–mm square plastic dishes with covers
24 × 60–mm coverslips
20-W fluorescent light
60° to 65°C water bath

CAUTION: Hoechst 33258 is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 5: Visualization of BrdU Replication Banding by Heat Treatment and Giemsa Staining

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Metaphase slides of BrdU-substituted chromosomes (preceding basic or alternate protocols)
1.0 M sodium phosphate buffer, pH 8.0 (see recipe)
4% Giemsa staining solution (see recipe)
87° to 90°C water bath

Alternate Protocol 4: B-Pulse Replication Banding for Fibroblasts

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Air-dried slides of metaphase chromosomes aged 1 week at room temperature (unit 4.1)
0.2 M HCl
5% (w/v) barium hydroxide [Ba(OH)₂; see recipe], 50°C
2× SSC (appendix 2D), 60°C
4% Giemsa staining solution in Gurrs buffer (see recipe)
Gurrs buffer, pH 6.8: dissolve Gurrs pH 6.8 buffer tablets (Bio/medical Specialties) in water according to manufacturer's instructions

Glass and polyethylene Coplin jars
50° and 60°C water baths

Support Protocol 1: Aging Slides with Heat

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Air-dried slides of metaphase chromosomes (<7 days old, unheated; unit 4.1)
2% gelatin solution (see recipe)
50% silver nitrate solution (see recipe)
3% (v/v) acetic acid (3 ml in 97 ml H₂O; prepare fresh)
65°C slide warmer or drying oven

NOTE: Silver nitrate is a protein stain and will stain skin and clothing. Wear gloves and lab coat.

Basic Protocol: Distamycin-DAPI Staining

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Air-dried slides of metaphase chromosomes (unit 4.1)
McIlvaine buffer, pH 7.0 and 7.5 (see recipe)
Distamycin A staining solution (see recipe)
DAPI staining solution (see recipe)
Immersion oil, low fluorescence

Humidified chamber (e.g., petri dish with moist paper towel)
Coverslips, no. 0 or no. 1

Alternate Protocol 6: Hoechst 33258–Distamycin Staining

Additional Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

PBS (appendix 2D)
Hoechst 33258 staining solution (see recipe)
Mounting buffer: 1:1 (v/v) glycerol/McIlvaine buffer, pH 7.5

Support Protocol 6: Destaining

Materials

For common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.

Stained slides
Xylene or Hemo-De (Fisher)
1:1 (v/v) xylene/ethanol (if xylene rather than Hemo-De is used)
95% ethanol
Fixative: 3:1 (v/v) methanol/acetic acid
Methanol (reagent grade)

CAUTION: Xylene is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Looking for materials? Suppliers Guide

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Figures

Figure 4.2.1

Q-banded metaphase spread from a phenotypically normal human male with an additional chromosome that is an isochromosome for the short arm of 15. (Material in Figs. 4.2.6, 4.2.7, and 4.2.8 comes from the same individual.) Chromosomes that can show Q-band polymorphisms are numbered. In this individual, there are moderate Q-bright bands on both copies of chromosome 3, Q-bright satellites on both copies of chromosome 13 and a Q-bright polymorphism on one 13 long arm (arrowhead), Q-bright satellites on one copy of chromosome 14 (the one in the satellite association with chromosome 21), Q-bright satellites on both ends of the isochromosome 15, and moderately bright satellites on one copy each of chromosomes 21 and 22. The Y chromosome shows bright fluorescence on the distal long arm and a Y-body is visible in the interphase nucleus (larger arrowhead). It is interesting that the i(15p) does not share polymorphisms with either of the normal 15s, suggesting it arose from different chromosomes 15. This individual's father also had the i(15p), indicating that it arose in an earlier generation in this family.

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Figure 4.2.2

G-banded metaphase spread from a phenotypically normal 46,XY male. X and Y chromosomes indicated by arrows. Chromosomes are stained with Giemsa.

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Figure 4.2.3

RHG R-banded metaphase spread from a phenotypically normal 46,XY male.

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Figure 4.2.4

CA3/DA R-banded metaphase spread from a phenotypically normal 46,XY male.

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Figure 4.2.5

(A) Replication-banded metaphase spread from a female patient with a translocation between the X chromosome and chromosome 8. The two translocation products of the t(X;8)(q13;p23) are indicated by small arrows. B-pulse BrdU substitution followed by visualization with Giemsa shows that the normal X chromosome (indicated by large arrow) is late-replicating as revealed by pale staining. Note R-banding-like pattern on other chromosomes. (B) Replication-banded metaphase spread from a female patient with an inversion of the X chromosome [inv(X)(p22q26), indicated by a small arrow]. T-pulse BrdU substitution followed by visualization with Giemsa shows that the normal X chromosome (indicated by large arrow) is late-replicating as revealed by dark staining. Note G-banding-like pattern on other chromosomes.

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Figure 4.2.6

C-banded metaphase spreads. (A) A 46,XX, phenotypically normal female with common inversion of the centromeric heterochromatin on chromosome 9. (B) Phenotypically normal male with a 47,XY,+i(15p) karyotype. Note prominent staining near all centromeres and larger blocks of staining near centromeres of chromosomes 1, 9, and 16, and at the distal portion of the Y chromosome (the region that shows bright Q-banding).

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Figure 4.2.7

NOR-stained metaphase spread. Sample from a phenotypically normal male with 47,XY,+i(15p) karyotype with NOR staining done after Q-banding for chromosome identification. In this individual, only 6 of the 10 normal acrocentric chromosomes showed NOR staining (chromosomes 13, 14, 15, 21, and 22), as well as both ends of the small isochromosome.

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Figure 4.2.8

Distamycin-DAPI staining of two metaphase spreads from a phenotypically normal 47,XY,i(15p) male. This patient's cells show prominent staining of centromeric heterochromatin of chromosomes 1, 9, 15, 16, and Y. This staining confirmed that the marker chromosome that was NOR-positive was derived from chromosome 15. (A) Increased distamycin staining to highlight heterochromatin. (B) Decreased distamycin staining to visualize chromosome banding.

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Literature Cited

Literature Cited for Q-Banding
	Caspersson, T., Farber, S., Foley, G.F., Kudynowski, J., Modest, E.J., Simonsson, E., Wagh, U., and Zech, L. 1968. Chemical differentiation along metaphase chromosomes. Exp. Cell Res. 49:219-222.
	Caspersson, T., Zech, L., and Johansson, C. 1970. Analysis of human metaphase chromosomes by aid of DNA binding fluorescent agents. Exp. Cell Res. 62:490-492.
	Dev, V.G., Warburton, D., Miller, O.J., Miller, D.A., Erlanger, B.F., and Beiser, S.M. 1972. Consistent pattern of banding of anti-adenosine antibodies to human metaphase chromosomes. Exp. Cell Res. 74:288-293.
	Distèche, C. and Bontemps, J. 1974. Chromosome regions containing DNAs of known base composition specifically evidenced by 2,7-di-t-butyl proflavin. Chromosoma 47:263-281.
	Distèche, C., Bontemps, J., Houssien, C., Frédérec, J., and Fredericq, E. 1980. Quantitative analysis of fluorescence profiles of chromosomes. Exp. Cell Res. 125:251-264.
	Korenberg, J.R. and Rykowski, M.C. 1988. Human genome organization: Alu, Lines, and the molecular structure of metaphase chromosome bands. Cell 53:391-400.
	Manuelidis, L. and Ward, D.C. 1984. Chromosomal and nuclear distribution of the Hin dIII 1.9 kb human DNA repeat segment. Chromosoma 91:28-38.
	Pachmann, U. and Rigler, R. 1972. Quantum yield of acridines interacting with DNA of defined base sequence. A basis for the explanation of acridine bands in chromosomes. Exp. Cell Res. 72:602-608.
	Schreck, R.R., Warburton, D., Miller, O.J., Beiser, S.M., and Erlanger, B.F. 1973. Chromosome structure as revealed by a combined chemical and immunochemical procedure. Proc. Natl. Acad. Sci. U.S.A. 70:804-807.
	Schreck, R.R., Erlanger, B.F., and Miller, O.J. 1974. The use of anti-nucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation. Exp. Cell Res. 88:31-39.
	Weisblum, B. and deHaseth, P.L. 1972. Quinacrine, a chemical stain specific for deoxyadenylate-deoxythymilate rich regions in DNA. Proc. Natl. Acad. Sci. U.S.A. 69:629-632.
	Weisblum, B. 1973. Fluorescent probes of chromosomal DNA structure: Three classes of acridines. Cold Spring Harbor Symp. Quant. Biol. 38:441-449.
Key Reference for Q-Banding
	Latt, S.A. 1976. Optical studies of metaphase chromosome organization. Annu. Rev. Biophys. Bioeng. 5:1-37.
During the 1970s, a number of review articles appeared that attempted to summarize all of the new developments in the field of cytogenetics. This article, with a 398-item reference list, covers a variety of banding methods with detailed explanations of their underlying biochemistry. This review discusses Q-banding as well as C-banding, G-banding, R-banding, and the use of BrdU incorporation for chromosome labeling.
Literature Cited for G-Banding
	Comings, D.E. 1978. Mechanisms of chromosome banding and implications for chromosome structure. Annu. Rev. Genet. 12:25-46.
	Drets, M.E. and Shaw, M.W. 1971. Specific banding patterns of human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 68:2073-2077.
	Seabright, M. 1971. A rapid banding technique for human chromosomes. Lancet II:971-972.
	Sumner, A.T. 1990. Chromosome Banding. Unwin Hyman, London.
	Sumner, A.T., Evans, H.J., and Buckland, R.A. 1971. New techniques for distinguishing between human chromosomes. Nature New Biol. 232:31-32.
	van Duijn, P., van Prooijen-Knegt, A.C., and van der Ploeg, M. 1985. Involvement of nucleosomes in Giemsa staining of chromosomes. A new hypothesis on the banding mechanism. Histochemistry 82:363-376.
	Wang, H.C. and Federoff, S. 1972. Banding human chromosomes treated with trypsin. Nature New Biol. 235:52-53.
Key References for G-Banding
	Comings, 1978. See above.
Review article with good discussions of possible underlying biochemical mechanisms of G-banding in relationship to chromosome structure.
	Comings, D.E., Avelino, E., Harris, D.C., and Wyandt, H.E. 1973. The mechanism of C- and G-banding of chromosomes. Exp. Cell Res. 77:469-493.
	Sumner, 1990. See above.
Good discussions of possible mechanisms of G-banding.
	Sumner, A.T. and Evans, H.J. 1973. Mechanism involved in the banding of chromosomes with quinicrine and Giemsa. II. The interaction of the dyes with the chromosomal components. Exp. Cell Res. 81:223-236.
Effects of the pretreatments and function of the Giemsa stain.
	van Duijn, et al. 1985. See above.
Presents a complex analysis of various steps in the G-banding process and how these steps may be influenced by nucleosome structure of chromosomes.
Literature Cited for R-Banding
	Latt, S.A., Jurgens, L.A., Matthews, D.J., Gustashaw, K.M., and Sahar, E. 1980. Energy transfer-enhanced chromosome banding. Cancer Genet. Cytogenet. 1:187-196.
	Sahar, E. and Latt, S.A. 1978. Enhancement of banding patterns in human metaphase chromosomes by energy transfer. Proc. Natl. Acad. Sci. U.S.A. 75:5650-5654.
	Schweitzer, D. 1981. Counterstain enhanced chromosome banding. Hum. Genet. 57:1-14.
	Sehested, J. 1974. A simple method of R-banding of human chromosomes, showing a pH dependent connection between R- and G-bands. Humangenetik 21:55-58.
	Sumner, A.T. 1990. Chromosome Banding. Unwin Hyman, London.
Key References for R-Banding
	Latt et al., 1980. See above.
	Schweitzer et al. See above.
These two articles review the mechanisms of double-dye staining.
Literature Cited for Replication Banding
	Gratzner, H., Ingram, D., Lief, R.C., and Castro, A. 1975. The use of antibody specific for bromodeoxyuridine for immunofluorescent determination of DNA replication in single cells and chromosomes. Exp. Cell Res. 95:88-94.
	Korenberg, J. and Freedlander, E. 1974. Giemsa technique for detection of sister chromatid exchanges. Chromosoma 48:355-360.
	Latt, S.A. 1973. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. U.S.A. 70:3395-3399.
	Latt, S.A., Willard, H.F., and Gerald, P.S. 1976. BrdU-33258 Hoechst analysis of DNA replication in human lymphocytes with supernumerary or structurally abnormal X chromosomes. Chromosoma 57:135-153.
	Lemieux, N., Dutrillaux, B., and Viegas-Pequignot, E. 1992. A simple method for simultaneous R- or G-banding and fluorescence in situ hybridization of small single-copy genes. Cytogenet. Cell Genet. 59:311-312.
	Perry, P. and Wolff, S. 1974. New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158.
Key Reference for Replication Banding
	Latt, S.A. 1976. Optical studies of metaphase chromosome organization. Annu. Rev. Biophys. Bioeng. 5:1-37.
An excellent review that covers the principles of replication banding.
Literature Cited for C-Banding
	Comings, D.E., Avelino, E., Okada, T.A., and Wyandt, H.E. 1973. The mechanism of C- and G-banding of chromosomes. Exp. Cell Res. 77:469-493.
	Holmquist, G. 1979. The mechanism of C-banding: Depurination and -elimination. Chromosoma 72:203-224.
	Pardue, M.L. and Gall, J.G. 1970. Chromosomal localization of mouse satellite DNA. Science 168:1356-1358.
	Pathak, S. and Arrighi, F.E. 1973. Loss of DNA following C-banding procedures. Cytogenet. Cell Genet. 12:414-422.
Key References for C-Banding
	Sumner, A.T. 1990. Chromosome Banding. Unwin Hyman, London.
	Holmquist, G. 1979. See above.
These references provide detailed discussion of the mechanism of C-banding.
Literature Cited for NOR Staining
	Goodpasture, C. and Bloom, S.E. 1975. Visualization of nucleolus organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37-50.
	Goodpasture, C., Bloom, S.E., Hsu, T.C., and Arrighi, F.E. 1976. Human nucleolus organizers: The satellites or the stalks. Am. J. Hum. Genet. 28:559-566.
	Henderson, A.S., Warburton, D., and Atwood, K.C. 1972. Location of ribosomal DNA in the human chromosome complement. Proc. Natl. Acad. Sci. U.S.A. 69:3394-3398.
	Howell, W.M. and Black, D.A. 1980. Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: A 1-step method. Experientia 36:1014-1015.
	Howell, W.M., Denton, T.E., and Diamand, J.R. 1975. Differential staining of the satellite regions of human acrocentric chromosomes. Experientia 31:260-262.
	Mikelsaar, A-V., Schwarzacher, H.G., Schnedl, W., and Wagenbichler, P. 1977. Inheritance of Ag-stainability of nucleolus organizer regions. Hum. Genet. 38:183-188.
	Miller, D.A., Dev, V.G., Tantravahi, R., and Miller, O.J. 1976. Suppression of human nucleolus organizer activity in mouse-human hybrid cells. Exp. Cell Res. 101:235-243.
	Miller, O.J., Miller, D.A., Dev, V.G., Tantravahi, R., and Croce, C.M. 1976. Expression of human somatic cell hybrids. Proc. Natl. Acad. Sci. U.S.A. 73:4531-4535.
Key Reference for NOR Staining
	Sumner, A.T. 1990. Chromosome Banding. Unwin Hyman, London.
Contains a good discussion of the methodology and basis of silver staining.
Literature Cited for Distamycin/DAPI Staining
	Buckton, K.E., Spowart, G., Newton, M.S., and Evans, H.J. 1985. Forty-four probands with an additional “marker” chromosome. Hum. Genet. 69:353-370.
	Coll, M., Frederick, C.A., Wang, A.H-J., and Rich, A. 1987. A bifurcated hydrogen bonded conformation in the d(A-T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin. Proc. Natl. Acad. Sci. U.S.A. 84:8385-8389.
	Miller, O.J., Schnedl, W., Allen, J., and Erlanger, B.F. 1974. 5-Methylcytosine localized in mammalian constitutive heterochromatin. Nature 251:636-637.
	Sahar, E. and Latt, S.A. 1980. Energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes. Chromosoma 79:1-28.
	Schreck, R.R., Breg, W.R., Erlanger, B.F., and Miller, O.J. 1977. Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15. Hum. Genet. 36:1-12.
	Schweizer, D., Ambros, P., and Andrle, M. 1978. Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A. Exp. Cell Res. 111:327-332.
Key References for Distamycin/DAPI Staining
	Sahar and Latt, 1980. See above.
	Schweizer, D. 1981. Counterstain-enhanced chromosome banding. Hum. Genet. 57:1-14.
Two excellent articles explaining dye interactions that lead to chromosome banding, including those between distamycin A and DAPI.