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Karyotype Analysis and Chromosome Banding

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Karyotype Analysis and Chromosome Banding Karyotype Analysis and Secondary article Chromosome Banding Article Contents . Methods of Chromosome Banding Wendy A Bickmore, MRC Human Genetics Unit, Edinburgh, Scotland, UK . Uses of Chromosome Banding . Number and Size of Bands A series of reproducible bands across metaphase chromosomes can be revealed by some . Basis for G-/R-banding treatments. These chromosome bands not only allow the identification of normal and . Bands Reflect the Domain Organization of the Genome abnormal chromosomes but they also tell us about fundamental aspects of the chromatin . Extreme Chromosome Bands structure and compartmentalization of the genome. Methods of Chromosome Banding segments. This has had an invaluable impact on human Nearly all methods of chromosome banding rely on genetics and medicine and the power of this approach has harvesting chromosomes in mitosis. This is usually been augmented by combining cytogenetics with fluores- achieved by treating cells with tubulin inhibitors, such as cence in situ hybridization (FISH). The detection of colchicine or demecolcine (colcemid), that depolymerize chromosome deletions associated with disorders, very the mitotic spindle and so arrest the cell at this stage. often contiguous gene syndromes, provided some of the Excessively long incubations with Colcemid result in first disease gene localizations in humans. Similarly, overcondensed chromosomes that band poorly and more- translocations have been important in pinpointing the over some cell types, especially those from the mouse, location of disease-associated genes and the characteristic eventually escape the Colcemid block and proceed through translocations associated with some leukaemias is impor- the cell cycle. tant, not only for understanding the molecular basis of Chromosome banding methods are either based on these cancers, but also for their diagnosis and prognosis. staining chromosomes with a dye or on assaying for a One of the best examples of this is the translocation particular function. The most common methods of dye- between human chromosomes 9 and 22 – t(9;22)(q34:q11) based chromosome banding are G-(Giemsa), R-(reverse), – or the Philadelphia chromosome diagnostic of chronic C-(centromere) and Q-(quinacrine) banding. Bands that myelogenous leukaemia (CML). show strong staining are referred to as positive bands; Comparisons of chromosome banding patterns can weakly staining bands are negative bands. However the confirm evolutionary relationships between species and staining patterns are not black and white, different bands also reveal changes in karyotype that may have been stain to different intensities (Francke, 1994). G-positive important in speciation. The banding patterns of human, bands are usually just called G-bands and likewise for R- gorilla and chimpanzee chromosomes are almost identical, positive (R-) bands. Positive C-bands contain constitutive though human chromosome 2 is the result of a fusion heterochromatin. Q-bands are considered equivalent to G- between two great ape chromosomes. There are also bands. extensive similarities between human chromosome bands The most widely used function-based banding method is and those of lower primates. replication banding and is based on the fact that different bands replicate their DNA at different times during S phase of the cell cycle. Generally, R-band DNA is replicated earlier than G-bands (Dutrillaux et al., 1976). G-bands also Number and Size of Bands correspond to the condensed chromomeres of meiotic chromosomes and R-bands to the interchromomeric Idealized diagrams (ideograms) of G-banded chromo- regions. somes are published as standard reference points for chromosome banding. The G-bands are usually portrayed in black and the R-bands in white. Bands are numbered consecutively away from the centromere on both the short Uses of Chromosome Banding (p) and long (q) arms (Figure 1). The total number of bands or ‘resolution’ in the human karyotype depends on how G-and R-bandingare the most commonly used techniques condensed the chromosomes are, and at what stage of for chromosome identification (karyotyping) and for mitosis they are in. A 350-band resolution corresponds to identifying abnormalities of chromosome number, trans- chromosomes late in metaphase. High-resolution ideo- locations of material from one chromosome to another, grams (approximately 1250–2000 bands) have also been and deletions, inversions or amplifications of chromosome produced for human chromosomes in mid-prophase ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Karyotype Analysis and Chromosome Banding 15.5 15.5 stain the under-denatured GC-rich regions. T-banding 15.4 15.4 identifies a subset of R-bands – the most intensely staining 15 15.3 15.3 15.2 15.2 ones – by employing either a more severe heat treatment 15.1 15.1 than R-banding. It is thought to identify the GC-richest R- 14.3 bands, of which approximately half occur at telomeres in 14 14 p p p 14.2 the human genome, hence the name. 14.1 The need to combine chromosome banding with 13 13 13 fluorescence in situ hybridization has meant that banding 12 12 12 techniques using fluorescent dyes has become more 11.2 11.2 11.2 popular. Q-banding involves staining with quinacrine 11.12 11.12 which reacts specifically with certain bases. Quinacrine 11.1 11.11 11.11 11 11 11 intercalates into chromosomal DNA irrespective of 12.1 12 12 12.2 sequence, but fluoresces brighter in regions of AT-rich 12.3 DNA. There are a number of other molecules whose 13.1 13.1 13.2 13.2 fluorescence is influenced by the base composition of the 13 13.3 13.3 13.4 13.4 DNA to which they are bound. In addition to quinacrine, 13.5 13.5 other commonly used fluorochromes with a specificity for 14.1 14.1 14 14.2 14.2 AT-rich DNA include Hoechst 33258, DAPI (4’-6- 14.3 14.3 diamidino-2-phenylindole) and daunomycin. The fluores- q 21 q 21 q 21 22.1 22.1 cence of Hoechst and DAPI is not quenched by guanine 22 22.2 22.2 and so they give less distinct bands than those produced by 22.3 22.3 quinacrine; however, daunomycin fluorescence is greatly 23.1 23.1 23.2 23.2 quenched by DNA with a GC content of 4 32%. DAPI 23 23.3 23.3 staining has the advantage that it is very resistant to fading and that its excitation and emission spectra are compatible 24.1 24 24 24.2 with reporter molecules and filters commonly used in FISH 24.3 25 25 25 (Figure 2). Fluorochromes with a preference for GC-rich DNA Figure 1 G-band ideograms of human chromosome 11 at (from left to include chromomycin and 7-amino actinomycin D. These right) 350, 550 and 850 band resolution. dyes give an R-band-like pattern. (Yunis, 1981). When a low-resolution band is subdivided, the number of each subband is placed behind a decimal Bands Reflect the Domain Organization point following the first band designation. For example the of the Genome most distal low-resolution band on the short arm of human chromosome 11 (11p15) can be subdivided into bands Techniques used to reveal chromosome bands enhance an 11p15.1, 11p15.2, 11p15.3, 11p15.4 and 11p15.5 at higher inherent pattern of chromosome organization. A chromo- resolution (Figure 1). A 2000-band resolution chromosome some band is a manifestation of a chromatin domain with band may contain 1.5 Mb of DNA, while a 300-band functional and structural characteristics that are homo- resolution band will contain 7–10 Mb of DNA. A skilled geneous and distinctive over a long enough stretch to be cytogeneticist may be able to spot a deletion of 5–10 Mb of seen down the microscope. G-and R-bandingreflect DNA depending on its location, but at a molecular level the differences in chromatin structure and base composition human genome probably comprises 4 2000 ‘bands’. between different regions of the genome. Fluorochrome banding also reflects variation in base composition along chromosome length. A rather different sort of banding is that seen after Basis for G-/R-banding staining of polytenized (interphase) chromosomes from some tissues of Dipteran insects. The basis for this banding G-banding involves staining protease-treated chromo- is not well understood but arises through the alignment, in somes with Giemsa dye and is thought to result from register, of many thousands of copies of the chromosome. interactions of both DNA and protein with the thiazine and eosin components of the stain. The most common R- Patterns in DNA sequence banding method involves heat denaturing chromosomes in hot acidic saline followed by Giemsa staining. This method Banding patterns can arise as a consequence of differences is thought to preferentially denature AT-rich DNA and to in the DNA sequence along chromosomes. R-and 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Karyotype Analysis and Chromosome Banding Figure 2 DAPI-stained chromosomes from mouse embryonic stem cells (male). The staining approximates to a G-band pattern, with the C- and G-bands stained more intensely by DAPI than the R-bands. The constitutive heterochromatin around the centromeres of these acrocentric chromosomes stains very intensely with DAPI. G-banding patterns are revealed on human chromosomes highlighting T-bands (Saccone et al., 1993). Molecular by FISH with Alu and LI interspersed repeats, respectively analyses on a finer scale reveal that, although there is a (Korenberg and Rykowski, 1988). A similar distribution general tendency for G-bands to be quite AT-rich, R-bands has also been reported for SINEs (short interspersed can contain both AT-rich isochores and GC-rich iso- elements) and LINEs (long interspersed elements) on chores. mouse chromosomes. However, molecular studies of the A fraction of DNA (CpG islands) that contains the 5’ human genome including sequencing show that both SINE ends of approximately 50% of mammalian genes also and LINE repeats can be found in close proximity to each produces a T-and R-bandingpattern on chromosomes other.
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