Metaphase chromosomes banding

Korenberg J. and Rikowski M. (1988). Human molecular organization alu, Lines, and the molecular structure of metaphase chromosome bands. Cell 53 391-400. 

Chromosomes are spread on the slide as metaphase chromosomes, stained with Giemsa solution to obtain G-banded patterns, and after appropriate washes and treatments, are ready for ISH. 

During mitosis, aU the DNA is highly condensed to allow separation of the sister chromatids. This is the only time in the ceE cycle when the chromosome structure is visible. Chromosome abnormalities may be assessed on mitotic chromosomes by karyotype analysis (metaphase chromosomes) and by banding techniques (prophase or prometaphase), which identify aneu-ploidy, translocations, deletions, inversions, and duplications. 

Y. Saitoh, and U. K. Laemmli, From the chromosomal loops and the scaffold to the classic bands of metaphase chromosomes. Cold Spring Harb. Symp. Quant. Biol. 58, 755-765 (1993). A. S. Belmont, and K. Bruce, Visualization of G1 chromosomes A folded, twisted, supercoiled chromonema model of interphase chromatid structure. J. Mol. Biol. 127, 287-302 (1994). 

Bhatt, B, Bums, J, Flannery, D., and McGee, J. O D. (1988) Direct visualization of single-copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization. Nucleic Acids Res 16,3951—3961... 

Figure 19.1 During the metaphase of cell division, a chromosome becomes two sister chromatids attached at the centromere. Chromosome banding exemplified using human chromosome 17.

Another property of such dyes is observed when they are used to stain metaphase chromosomes. When these dyes bind to chromosomal DNA, some parts of the chromosome fluoresce stronger than others giving a typical and reproducible fluorescence pattern. These fluorescence are used for chromosome analysis and identification. Andreoni et al.48 50) investigated the fluorescence decay of in band and out of band parts in Vicia fabia chromosomes stained with quinacrine mustards. They used a microbeam system (Chapter 3) with a nitrogen laser pumped dye laser. Their results seem to suggest that the band considered was to be attributed mainly to a higher dye concentration inside than outside the band. 

For mapping, BrdU is added to enhance chromosome elongation and banding for metaphase chromosomes and to help distinguish G1 from S and G2 phase nuclei in the case of interphase chromosomes. Treatment of the cells for a few minutes with hypotonic solutions to make them swell and exposure to low temperatures, thus interfering with the stability of spindle fibres, can improve the preparations. 

Metaphase chromosomes are often G-banded, karyotyped and destained before ISH, although there is some loss of hybridization signal (Table 11.7). The tissues or cells should be actively dividing (hematopoietic tissue, primary cell culture). Mitogen stimulation often has a different effectiveness for different tissues or for similar tissues from different animal species (Davisson and Akeson, 1987). 

A EXPERIMENTAL FIGURE 10-5 Simple-sequence DNAs are useful chromosomal markers. Human metaphase chromosomes stained with a fluorescent dye were hybridized in situ with a particuiar simpie-sequence DNA iabeied with a fluorescent biotin derivative. When viewed under the appropriate wavelength of light, the DNA appears red and the hybridized simple-sequence DNA appears as a yellow band on chromosome 16, thus locating this particular simple sequence to one site in the genome. [See R. K. Moyzis et at, 1987, Chromosoma 95 378 courtesy of R. K. Moyzis.]... 

During Metaphase, Chromosomes Can Be Distinguished by Banding Patterns and Chromosome Painting... 

G bands are produced when metaphase chromosomes are subjected briefly to mild heat or proteolysis and then stained with Giemsa reagent, a permanent DNA dye (Figure... 

Banding analysis and chromosome painting, a more precise method, are used to identify the different human metaphase chromosomes and to detect translocations and deletions (see Figure 10-29). 

Metaphase chromosomes can be Identified by characteristic banding patterns. What are G bands and R bands What is chromosome painting, and how Is this technique useful ... 

Saccone S., De Sario A., Della Valle G., Bernardi G. (1992) The highest gene concentrations in the human genome are in T bands of metaphase chromosomes. Proc. Natl. Acad. Sci. USA 89 4913-4917... 

All Raman bands measured in DNA fibres or crystals appear in this chromosome Micro-Raman spectrum. In addition, typical vibrations of the protein component were observed (phenylalanine, tyrosine, S—S group and the amide I mode). Recently, Micro-SERS has been applied for the first time to investigate the chromosomes adsorbed at the silver electrode This Micro-SERS spectrum of Chinese hamster metaphase chromosomes shows a number of intense bands. The enhancement factor obtained was estimated to be about 100 for the 790 cm DNA backbone vibration. The most intense bands in this SERS spectrum are located at 730 cm " and 1330 cm and can be attributed to the adsorbed adenine base vibration of the DNA. The characteristic protein vibrations in the normal Raman spectrum are missing in the SERS spectrum. 

Saitoh, Y., and Laemmli, U. K. (1993). From the chromosomal loops and the scaffold to the classic bands of metaphase chromosomes. Cold Spring Harbor Symp. Quant. BioL 58, 755-765. 

Cytogenic Analysis. In cytogenic analysis, cells are harvested and processed to visualize chromosomes in mitotically active cells. The cells are arrested in metaphase, and chromosome bands are visualized... 

Stockert, J. C. Pinna-Senn, E. Bella, J. L. Lisanti, J. A. DNA-binding fluorochromes correlation between C-banding of mouse metaphase chromosomes and hydrogen bonding to adenine-thymine base pairs. Acta Histochem. 2005, 106, 413-420. 

Presently karyotypes of human metaphase chromosomes are used to detect genetic defects like deletions or translocations, where the chromosomes are treated by the trypsin-Giemsa protocol, to produce a typical banding pattern and imaged by optical microscopy. Because of the diffraction limit in optical microscopy, even the smallest visible band contains around 1 million base pairs. Improved resolution has been demonstrated using fluorescence NSOM on the treated chromosomes compared to conventional light microscopy. 

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