Chromosome Preparation and Banding

Charleen M Moore, University of Texas Health Science Center at San Antonio, Texas, USA Robert G Best, University of South Carolina School of Medicine, Columbia, South Carolina, USA
Reliable techniques have been developed to produce large numbers of mitotic cells and to collect them at metaphase in order to visualize individual chromosomes. After cell culture and spreading of the metaphases onto slides, the chromosomes are stained to produce unique banding patterns or to reveal specialized structures. Molecular techniques have been developed to identify submicroscopic rearrangements and to compare karyotypes of different species.

Secondary article
Article Contents
. Introduction . Chromosome Spreads . Classical Staining Methods . Standard Banding Methods . Advanced Banding Methods . Molecular Cytogenetics . Conclusions

Introduction
Chromosome preparation and banding can be considered an art as well as a science. Chromosomes are visualized individually only during mitosis, and therefore techniques have been developed to stimulate large numbers of cells to begin division through the use of mitogens such as phytohaemagglutinin and pokeweed and to collect the cells at metaphase using spindle inhibitors such as colcemid. Numerous methods are now available for identifying chromosomes and preparing karyotypes for clinical and research purposes, although the ability to analyse chromosomes is dependent on the length of the chromosomes and how well they are xed, spread and stained.

Chromosome Spreads
Visualization of human chromosomes in somatic cells requires that dividing cells be studied during mitosis. Some cells may, by chance, be caught in the metaphase or anaphase part of the cell cycle at the time that cells are prepared for study under the microscope. However, large numbers of metaphase cells can best be obtained by growing cells in culture, and adding spindle poisons such as colcemid to cell cultures during periods of active growth to arrest cells in metaphase. While the number of cells found in metaphase will increase as the length of exposure to the spindle poison increases, chromosome condensation also progresses with time. The optimal length of exposure to the spindle poison will be determined by the rate of cell division and the degree of condensation that is desired. Many cell types undergo growth and division spontaneously, but some cell types, such as peripheral lymphocytes, need to be stimulated into mitotic activity by the addition of mitogens at the time cell cultures are initiated. A variety of mitogens are available for use in lymphocyte

culture. The most commonly employed are phytohaemagglutinin (PHA) for stimulation of T cell lymphocytes, and pokeweed mitogen for the stimulation of B cell lymphocytes. Certain cytogenetic procedures are optimized when all of the cells in culture are synchronized in their mitotic cycle. This is achieved by adding chemical agents that block progression into S phase to an actively growing culture for 1620 h. Excess thymidine, or the DNA antimetabolites amethopterin, bromodeoxyuridine (BrdU) and uorodeoxyuridine are eective agents for synchronization of cell cultures. Release of the S phase block by resuspending cells in fresh medium is performed a few hours prior to harvest. Synchronization is critical in replication banding methods where chromosome identication is achieved by the incorporation of DNA base analogues such as BrdU. One key element in the preparation of analysable chromosome spreads is the degree of dispersion of the chromosomes on the microscope slide. Optimal dispersion is inuenced by several variables at the time of cell harvest. The ideal metaphase spread has all 46 chromosomes dispersed in the same optical eld under the microscope, with no overlapping chromosomes. The harvesting procedure involves centrifugation of cell suspensions into a cell pellet, treatment with a hypotonic salt solution, xation of the suspended cell pellet, and dropping of the cells onto glass slides. Each of the steps in the harvesting procedure may inuence the dispersion of the chromosomes on the slide. Time invested in optimizing the spreading of chromosomes and preparing good slide preparations can save countless hours in the analysis phase of a cytogenetic study. Treatment with a hypotonic salt solution just prior to harvest permits swelling of the nuclei. Incubation in a dilute KCl or sodium citrate solution for 1030 min generally achieves good spreading. Insucient hypotonic treatment results in chromosome spreads that are tightly
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Chromosome Preparation and Banding

knotted; individual chromosomes are dicult to virtually impossible to visualize. Over-treatment with hypotonic solution results in scattering of chromosomes, or rupture of the nuclei and loss of the chromosomes. Preservation of the cells is the nal step before the preparation of slides. Fixation with Carnoys solution, a mixture of methanol and glacial acetic acid, arrests the process of hypotonic swelling and all metabolic processes of the cells, and preserves cells in a stable state. Care must be exercised to suspend the cells in the cell pellet prior to and during xation to avoid clumping of cells and poor spreading. Three or more rounds of suspension in fresh Carnoys and centrifugation of cells into a pellet are usually employed to prepare cells for dropping onto slides. Slide making is not a science. Although careful attention to a number of variables certainly increases the chance of successful results, this aspect of cytogenetic technology is something of an art. Drops of xed cell suspension are placed onto glass slides and the xative is allowed to evaporate. Examination of the slide under a phase microscope while the xative is evaporating reveals the frenetic dancing of the xed cells until the liquid is nearly gone. Metaphase cells attach one by one onto the slide surface as the nal liquid disappears, and the chromosomes appear much like a ower in bloom as the nal traces of xative evaporate. As the slide dries completely, the chromosomes become set immovably on the glass slide. The rate at which the xative evaporates is critical to the nal dispersion of the chromosomes on the slide. Thus, humidity, temperature and the ow of air blown over the surface of the drying slide can be manipulated to produce optimal chromosome preparations.

defects and in dening chromosome structure such as the position of the centromeres and nucleolar organizing regions.

Standard Banding Methods

Q-Banding
In the late 1960s Caspersson postulated that dierences in DNA base composition might produce dierential intensity patterns along the length of chromosomes when uorescent DNA-binding dyes were applied to chromosome spreads, and thus the concept of chromosome banding was born. Fluorescent banding was demonstrated in plant chromosomes in 1968 using quinacrine mustard, and in 1971 the quinacrine (Q-) banding pattern for all 24 human chromosomes (22 autosomes, X, and Y) was reported. While the actual molecular basis for dierential quinacrine staining is not quite as Caspersson imagined, it became apparent that regions of the genome in which the bases adenine and thymine were relatively abundant (ATrich) tended to produce intense uorescence, while regions containing abundant guanine and cytosine residues (GCrich) uoresced more weakly. Most importantly, all 24 human chromosomes could be unequivocally identied for the rst time, and clinical cytogenetics studies for structural as well as numerical chromosome abnormalities became possible. Quinacrine banding is relatively simple to perform, although visualization of the uorescence pattern requires uorescence microscopy resources and a photomicroscope to capture the short-lived uorescence pattern on lm. Other uorescent stains produce similar patterns to that of quinacrine, including Hoescht 33258, DAPI (4,6-diamidino-2-phenylindole) and diimidazolinophenylindole (DIPI). All of these banding patterns are considered to be forms of Q-banding. Counterstaining of chromosomes with a second dye such as distamycin A or actinomycin D, or manipulation of pH, can enhance the sharpness and brightness of Q-bands.

Classical Staining Methods

A wide variety of stains are useful for visualizing chromosomes under the microscope. Classical cytological stains such as aceto-orcein, acetocarmine, gentian violet, and haematoxylin readily stain chromatin and are easy to visualize under the standard light microscope. While acetoorcein is noted to produce a crisp staining pattern that permits the study of chromosome morphology, unfortunately it is indelible and does not permit destaining and use of subsequent staining methods for banding. Other stains, such as Giemsa, Wright and Leishman stains can be readily removed with solvents, and are more often employed when unbanded preparations are under study. Chromosome arms, primary constrictions, satellites, stalks and fragile sites are readily recognizable with classical staining. Since the advent of chromosome banding methods, classical staining methods are rarely employed in the clinical analysis of human chromosomes. The chief applications currently for classical staining are in the study of breakage in chromosomes from ageing, clastogens, or DNA repair
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G-Banding
Soon after the discovery of Q-banding, a second method, Giemsa (G-) banding, was introduced that utilized the common Giemsa stain following various chemical and enzymatic treatments of the chromosome preparations (Figure 1). This method oered the advantage of producing permanent slides that can be studied under a standard light microscope. The pattern of staining in G-banded preparations is quite similar to that in Q-banded preparations (i.e. intense Giemsa-stained regions correlate with intense Qbanded uorescent regions). G-Banding is most consistently produced by pretreatment of chromosomes with trypsin before staining with Giemsa. Other stains, such as

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Chromosome Preparation and Banding

Figure 1 G-Banding (a) Normal human male metaphase spread showing 46 human chromosomes. G-Bands were produced by treatment with trypsin followed by staining with Giemsa. (b) The same metaphase arranged in standard karyotype format.

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Chromosome Preparation and Banding

Wright stain and Leishman stain can be used eectively in the place of Giemsa to produce a pattern identical to that obtained with Giemsa, but with slightly dierent contrasting properties. A standard procedure for clinical study of chromosomes is to photograph (or digitize onto computer disk) the entire metaphase spread, cut out the individual chromosomes (actually or electronically), and arrange the chromosomes in a standard karyotype where both homologues of each chromosome pair are placed side by side in numerical order. Arranged in this manner, careful band-by-band analysis can be performed, which permits identication of even relatively subtle changes in banding patterns caused by structural chromosome abnormalities. Bands that are dark with G-banding (and bright with Q-banding) generally correspond to late-replicating regions of the genome. These bands tend to contain relatively few active genes. Pale bands typically correspond to earlier-replicating regions and are more gene-rich than are light bands.

present, and for the study of chromosomal polymorphisms in the population. The short arms and satellites of acrocentric chromosomes, pericentric heterochromatin, and much of the long arm of the Y-chromosome are all Cband-positive, contain no active genes, and show variations in size in normal individuals.

Advanced Banding Methods

High-resolution banding
A variety of techniques have been described with more specialized applications than the standard banding techniques. These methods permit more intense scrutiny of various aspects of the human karyotype. High-resolution banding techniques are designed to allow more detailed analysis of chromosomal bands across the entire karyotype, while other specialized techniques focus on specic areas or regions of individual chromosomes. The number of bands that is discernible in a single metaphase chromosome spread may vary from under 300 to approximately 1400, counting bands from only one homologue of each chromosome pair and the Y chromosome when present. The number of identiable bands in any spread is related to the degree to which the chromosomes are permitted to condense before harvest, the cell type, and the method of banding employed. The degree of diculty in completing an analysis of the karyotype is directly related to the number of bands that can be identied. Suspected aneuploidy (e.g. trisomy 21) can readily be evaluated at fairly low band resolution levels (i.e. 350550 bands), while suspicion of subtle deletions and other structural rearrangements requires higher band resolution levels (650 bands or more). High-resolution banding can be achieved by several methods. First, cells that have been xed in late prophase or early metaphase exhibit minimal chromatin condensation and maximal band resolution. Synchronization of cell cultures followed by relatively short exposures to colcemid produces cell preparations with a very low degree of chromosome condensation and thus a high band level. Similar results can be obtained by using a variety of additives to the culture that intercalate into or bind to the DNA molecule, inhibiting chromosome condensation in the process. Ethidium bromide, acridine orange and actinomycin D are frequently used in this manner. A third method of achieving high-resolution banding relies on the dierential uptake of DNA base analogues by early- versus late-replicating bands within the genome. This method, termed replication banding produces chromosome preparations with the highest band levels, as high as 1400 bands per haploid genome. Using replication banding, both R- and G-banded patterns can be produced. The pattern of banding is controlled by changing the timing of

R-Banding
A pattern that is approximately the opposite of G- or Qbanding can be produced by various means and is referred to as reverse (R-)banding. Fluorescent R-banding patterns are produced by dyes with GC base-pair anity such as chromomycin A3, olivomycin and mithramycin. Fluorescent R-banding patterns can often be enhanced by counterstaining with a second dye such as distamycin A, methyl green, actinomycin D or netropsin. R-Bands can also be produced by subjecting slides to high temperatures for several minutes followed by staining with Giemsa or acridine orange. R-Bands have the theoretical advantage of staining the gene-rich chromatin, thus enhancing the ability to visualize small structural rearrangements in the parts of the genome that are most likely to result in phenotypic abnormalities.

C-Banding
Noncoding constitutive heterochromatin, such as the repetitive DNA surrounding the centromeres of all of the chromosomes, replicates later in the cell cycle than other chromatin and exhibits special characteristics of stability under extreme conditions of heat and chemical exposure. This property of tightly condensed heterochromatin can be exploited to produce a unique banding pattern (Cbanding) in which the constitutive heterochromatin stains darkly and all other chromatin remains pale. C-Banding is produced by treatment of chromatin with acidic and then basic solutions followed by staining with Giemsa. CBanding is of limited use in the clinical laboratory and is primarily of value in the identication of the gene coding potential of various segments of the genome, especially when small marker chromosomes of unknown origin are
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Chromosome Preparation and Banding

the pulse addition of the DNA base analogue, BrdU, into growing, synchronized cultures. High-resolution banded metaphase spreads require optimal chromosome spreading if analysis is to be completed in any reasonable length of time. While these techniques are very sensitive for subtle chromosome rearrangements, they are generally reserved for use in clinical cases with a high suspicion of subtle chromosome abnormalities because of the intense labour involved in completing the analysis. Often, a clinical phenotype will suggest specic areas of the karyotype that should be studied with the detail available from high-resolution banding.

Restriction enzyme digestion

Variable patterns of chromatin staining can be observed in dierent patients when chromosomes are digested by certain restriction enzymes such as AluI, DdeI, HaeIII, HinfI, MboI, or RsaI. Most of the variability is found in regions of heterochromatin in the pericentromeric areas and on the short arms of the acrocentric chromosomes. These methods are useful in studying chromosome polymorphisms in the population, and more rarely for the purpose of identifying marker chromosomes and the parental origins of individual homologues.

Other specialized techniques Sister chromatid exchange

The two sister chromatids of a chromosome can also be dierentially stained by the addition of BrdU during cell culture and can reveal exchanges between the two chromatids (sister chromatid exchange, SCE). This requires two rounds of replication in BrdU because of the semiconservative nature of DNA replication. After a single cell division, uptake of BrdU (which replaces thymidine) results in homogeneous staining of both chromatids, with each double-stranded DNA molecule (one for each chromatid) containing one strand of the parental (unsubstituted) DNA and one strand of the BrdU-substituted DNA. Dierential staining of the two chromatids can be seen after a second cell division in the presence of BrdU. Here, the original parental strand of DNA (without BrdU incorporation) remains on one chromatid, paired with a newly synthesized BrdU-substituted strand, while the other chromatid has BrdU incorporated into both strands. Exposure of the chromosomes to the uorescent stain Hoechst 33258 and UV light causes loss of chromatin in the chromatid, which is composed of two BrdU-substituted DNA strands, with relatively light staining on exposure to Giemsa stain. The chromatid that has only a single BrdUsubstituted strand is more stable and loses less chromatin on exposure to the combination of stain and UV light, and thus stains more darkly with Giemsa stain. Exchanges between sister chromatids are evidenced by discontinuous light and dark staining regions along the length of the chromatids, one sister chromatid showing the opposite staining pattern from the other. SCE occurs naturally at a rate of 610 SCE/cell in normal cells grown in BrdU. This method is used as a diagnostic test for Bloom syndrome in the clinical laboratory, where SCE frequencies are extraordinarily high owing to inherent chromosomal instability. SCE is also used as an in vitro genotoxicity assay in the toxicology laboratory to identify chemical agents with genotoxic potential. A variety of specialized techniques have been described that dierentially stain chromosome telomeres (T-banding), the pericentromeric region of chromosome 9 (G11banding), the short arm of chromosome 15 (distamycin/ DAPI banding), centromeric dots (Cd-banding), and active nucleolar organizing regions (NOR or silver staining). Each of these methods, though very narrow in their application, may shed signicant light on a variety of cytogenetic abnormalities and normal polymorphisms. However, many of these methods have now been displaced in the clinical cytogenetics laboratory by molecular cytogenetic methods, which permit conclusive identication of most regions of the genome.

Molecular Cytogenetics
Fluorescence in situ hybridization
A wide variety of molecular cytogenetic methods have been described in which labelled DNA probes for specic sequences in the human genome can be hybridized to human chromosomes to locate and enumerate the DNA sequences of interest. The label used is typically uorescent, and thus uorescence in situ hybridization (FISH) is the standard procedure employed. Other labelling methods, both isotopic and nonisotopic, have also been employed for the same purpose. Dierently coloured uorochromes in the visible and infrared spectrum are available for use and allow the simultaneous detection of multiple probes, each with a unique colour. Two lters are needed for each uor to be visualized: an excitation lter that directs UV light toward the specimen within a range of wavelengths that causes the uor to uoresce, and a barrier lter that screens out extraneous light emitted from the specimen to permit only the colour of interest to be visualized. Unique repetitive DNA sequences in the a-satellite heterochromatin anking the centromere are present in most chromosomes, and FISH probes for these regions yield intensely bright signals that can be visualized in both
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Chromosome Preparation and Banding

interphase and metaphase cell preparations (Figure 2a). These probes are most useful for enumeration of individual chromosomes. Inclusion of interphase cells for study permits much larger sample sizes, allows for study of nondividing cell populations, and eliminates the culture time needed for mitotic preparations. Unique nonrepetitive sequences can also be identied with FISH probes. While the signals produced by cosmid FISH probes are smaller and less intense, distinct punctate signals can readily be identied on each chromatid of both homologues. This method can be used to localize

individual DNA sequences within the genome, and is especially valuable in identifying small deletions or duplications (which may be suspected on the basis of clinical phenotype) that are too small to be detected by conventional cytogenetic methods. Clinical microdeletion and microduplication syndromes that are dicult to identify by conventional cytogenetic methods are readily identied in the majority of cases by FISH (Figure 2b). Other probes have been particularly valuable in cancer cytogenetics of leukaemias and other neoplasias where specic chromosome rearrangements correlate with the

Figure 2 Molecular cytogenetic probes. (a) Normal human metaphase spread showing hybridization of the centromeric region of chromosomes 7 (green) and 8 (red) using a-satellite probes. Chromosomes counterstained in blue using DAPI. (b) Human male metaphase spread with deletion of the elastin (ELN) locus at the Williams syndrome critical region near the centromere on one homologue of chromosome 7. Cosmid probe for the ELN locus and a control probe are visible on the normal homologue (right); however, only the control probe can be seen on the deleted homologue (left). (c) Normal human metaphase spread with whole-chromosome paint probe for chromosome pair number 15 in red. (d) Cross-species colour banding (RXFISH1) on normal human male chromosomes arranged in standard karyotype format.

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Chromosome Preparation and Banding

type and severity of the cancer and may inuence the plan for treatment or therapy. Chromosome paints are libraries of DNA probes spanning an entire chromosome or chromosome arm that are unique to the chromosome in question. When labelled with a uorochrome, the probe libraries produce a signal only on the chromosome of interest (Figure 2c). These probes are useful for identifying the chromosomal origins of structurally abnormal chromosomes and markers.

Multicolour FISH
Three methods have been advanced that permit the simultaneous detection of all 24 human chromosomes: spectral karyotyping (SKY1), multiplex FISH (M-FISH), and cross-species colour banding (RX-FISH1). In SKY1 and M-FISH, a series of ve dyes are used to label each chromosome with a unique colour. A third method, RXFISH1, employs labelled probes obtained from a variety of primates for hybridization to human chromosomes. These produce a multicoloured banding pattern that, like G-banding, is unique for each chromosome (Figure 2d). Each of these techniques provides a useful tool for evaluating complex chromosomal abnormalities in humans, for rapidly constructing karyotypes for other species and for performing comparative genome mapping. (Note: SKY1 is a registered trademark of Applied Spectral Imaging, Inc., and RX-FISH1 is a registered trademark of Applied Imaging, Inc.)

Chromosomes were rst observed by uniformly staining chromatin with classic stains such as Giemsa. Now, each chromosome in the karyotype can be accurately identied using Q-, G- or R-banding to produce unique banding patterns with a total of up to 1400 bands per karyotype. Specic areas or structures such as centromeres and NORs can be identied through special staining techniques such as C-banding and silver staining. Sister chromatids can be dierentially stained through the incorporation of BrdU into the DNA during cell culture. Molecular cytogenetic analysis using techniques such as FISH can detect microdeletions and duplications that are not visible even with high-resolution banding. These techniques have had a dramatic eect on the clinical detection of many dierent syndromes. Through the use of FISH and other molecular techniques, chromosome number and specic DNA sequences can also be identied in nondividing cells. New techniques are being developed for clinical and research laboratories that will allow the simultaneous detection of all 24 human chromosomes and the evaluation of complex chromosome abnormalities. They also will provide the means for rapid comparative genome mapping between humans and other species and will facilitate investigation into the evolution of karyotypes of dierent species and comparison with the human genome.

Further Reading
Barch MJ, Knutsen T and Spurbeck JL (eds) (1997) The AGT Cytogenetics Laboratory Manual, 3rd edn. Philadelphia: LippincottRaven. Rooney DE and Czepulkowski BH (eds) (1992) Human Cytogenetics: A Practical Approach, vol. I, Constitutional Analysis, 2nd edn. Oxford: IRL Press. Rooney DE and Czepulkowski BH (eds) (1994) Human Cytogenetics. Essential Data. Chichester: Wiley. Sandberg A (1990) The Chromosomes in Human Cancer and Leukemia, 2nd edn. New York: Elsevier. Therman E and Susman M (1993) Human Chromosomes: Structure, Behavior, and Eects, 3rd edn. New York: Springer-Verlag. Verma RS and Babu A (1995) Human Chromosomes: Principles and Techniques, 2nd edn. New York: McGraw-Hill.

Conclusions
The improvement in preparation and identication of chromosomes and their component structures over the past few decades has been remarkable. Reliable techniques are now available for obtaining large numbers of cells in division, using mitogens to stimulate specic cell types. Chemicals that disrupt the spindle and swell the cells produce well-spread chromosomes that can be reliably counted and stained for various purposes.

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