1. Chromosome Preparation & Banding

2. Introduction
Chromosome preparation and banding can be considered an art as well as a science.
Chromosomes are visualized individually only during mitosis.
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.
The ability to analyse chromosomes is dependent on the length of the chromosomes and how well they are fixed, spread and stained.

3. Chromosome Spreads

4. Visualization of human chromosomes in somatic cells requires that dividing cells be studied during mitosis.
Adding spindle poisons such as colcemid to cell cultures during periods of active growth to arrest cells in metaphase.
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.

5. 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 ; by adding chemical agents that block progression into S phase to an actively growing culture for 16–20 h :
Excess thymidine, or the DNA antimetabolites amethopterin, bromodeoxyuridine (BrdU) and fluorodeoxyuridine .
Release of the S phase block by resuspending cells in fresh medium is performed a few hours prior to harvest.

6. One key element in the preparation of analyzable chromosome spreads is the degree of dispersion of the chromosomes on the microscope slide.
The ideal metaphase spread has all 46 chromosomes dispersed in the same optical field 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, fixation of the suspended cell pellet, and dropping of the cells onto glass slides.

7. 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 10–30 min generally achieves good spreading.
Insufficient hypotonic treatment results in chromosome spreads that are tightly knotted; individual chromosomes are difficult 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.

8. Preservation of the cells is the final step before the preparation of slides.
Fixation with Carnoy’s 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.
Three or more rounds of suspension in fresh Carnoy’s and centrifugation of cells into a pellet are usually employed.
Drops of fixed cell suspension are placed onto glass slides and the fixative is allowed to evaporate.
Metaphase cells attach one by one onto the slide surface as the final liquid disappears, and the chromosomes appear much like a flower in bloom as the final traces of fixative evaporate.

9. 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 aceto-orcein 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.

10. Chromosome arms, primary constrictions, satellites, stalks and fragile sites are readily recognizable with classical staining.
The chief applications currently for classical staining are in the study of breakage in chromosomes from ageing, clastogens, or DNA repair defects and in defining chromosome structure such as the position of the centromeres and nucleolar organizing regions.

11. Standard Banding Methods

12. Q-Banding
In the late 1960s Caspersson postulated that differences in DNA base composition might produce differential intensity patterns along the length of chromosomes when fluorescent 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.

13. It became apparent that regions of the genome in which the bases adenine and thymine were relatively abundant (A-T rich) tended to produce intense fluorescence, while regions containing abundant guanine and cytosine residues (G-C rich) fluoresced more weakly.
Quinacrine banding is relatively simple to perform, although visualization of the fluorescence pattern requires fluorescence microscopy resources and a photomicroscope to capture the short-lived fluorescence pattern on film.
Other fluorescent stains produce similar patterns to that of quinacrine, including Hoescht 33258, DAPI (4’,6’-diamidino-2-phenylindole) and diimidazolinophenylindole (DIPI).
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.

15. G-Banding
Giemsa (G-) banding, was introduced that utilized the common Giemsa stain following various chemical and enzymatic treatments of the chromosome preparations.
This method offered 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 Q banded fluorescent regions).
G-Banding is most consistently produced by pretreatment of chromosomes with trypsin before staining with Giemsa.

16. Other stains, such as Wright stain and Leishman stain can be used effectively in the place of Giemsa to produce a pattern identical to that obtained with Giemsa, but with slightly different contrasting properties.
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.

17. Staining with leishman
Staining with Giemsa
Staining with leishman

20. R-Banding
A pattern that is approximately the opposite of G- or Q banding 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 affinity 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.

21. 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.

23. 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 (C-banding).
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.

24. C-Banding is of limited use in the clinical laboratory and is primarily of value in the identification of the gene coding potential of various segments of the genome, especially when small marker chromosomes of unknown origin are 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 C-band-positive, contain no active genes, and show variations in size in normal individuals.

26. Conventional
G-banding
C-banding

27. Advanced Banding Methods

28. High-resolution banding
High-resolution banding techniques are designed to allow more detailed analysis of chromosomal bands across the entire karyotype, while other specialized techniques focus on specific 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.

29. The number of identifiable 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.
Suspected aneuploidy (e.g. trisomy 21) can readily be evaluated at fairly low band resolution levels (i.e. 350–550 bands).
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 fixed 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.

30. 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 relies on the differential uptake of DNAbase 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.
These techniques are very sensitive for subtle chromosome rearrangements.

31. Sister chromatid exchange
The two sister chromatids of a chromosome can also be differentially 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.
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.

32. Exposure of the chromosomes to the fluorescent stain Hoechst 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 BrdU-substituted 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.
SCE occurs naturally at a rate of 6–10 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.

33. Thanks for your attention
Saba Mardoukhi