Staining
The study of karyotypes is made possible by staining. Usually, a suitable dye, such as Giemsa, is applied after cells have been arrested during cell division by a solution of colchicine. For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture Sometimes observations may be made on non-dividing (interphase) cells. The sex of an unborn fetus can be determined by observation of interphase cells through amniotic centesis and Barr body. Variation is often found:
- 1. Between the sexes
- 2. Between the germ-line and soma (between gametes and the rest of the body)
- 3. Between members of a population (chromosome polymorphism)
- 4. Geographical variation between races
- 5. Mosaics or otherwise abnormal individuals.
The human karyotype
The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Normal karyotypes for females contain two X chromosomes and are denoted 44,XX; males have both an X and a Y chromosome denoted 44,XY. Any variation from the standard karyotype may lead to developmental abnormalities.
Types of banding
Cytogenetics employs several techniques to visualize different aspects of chromosomes:
- G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands – the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich. This method will normally produce 300-400 bands in a normal, human genome.
- R-banding is the reverse of G-banding (the R stands for “reverse”). The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).
- C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres. There fore useful for the identification of chromosomes based on the position of the centromere. The technique is not so useful as many chromosomes are similar in the position of their centromere.
- Q-banding is a fluorescent pattern in ultra violet light obtained using quinacrinemustard for staining. The pattern of bands is very similar to that seen in G-banding.
- T-banding: visualize telomeres.
- Silver staining/N banding: Silver nitrate stains the nucleolar organization region-associated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR.
- Other banding techniques : Other fluorescent dyes like Acridine orange can be used for the banding purpose.
In the “classic” (depicted) karyotype, a dye, often Giemsa (G-banding), less frequently Quinacrine, is used to stain bands on the chromosomes. Giemsa is specific for the phosphate groups of DNA. Quinacrine binds to the adeninethymine-rich regions. Each chromosome has a characteristic banding pattern that helps to identify them; both chromosomes in a pair will have the same banding pattern.
Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom. Some karyotypes call the short and long arms p and q, respectively. In addition, the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example, Cri du chat syndrome involves a deletion on the short arm of chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is deletion of 15.2, which is written as 46,XX,del(5)(p15.2)
Spectral karyotype (SKY technique)
Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes.
This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.
Digital karyotyping
Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. This method is also known as virtual karyotyping.
Human chromosomes are divided into 7 groups & sex chromosomes
Chromosome abnormalities
Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural, as in derivative chromosome, translocations, inversions, large-scale deletions or duplications. Numerical abnormalities, also known as aneuploidy, often occur as a result of nondisjunction during meiosis in the formation of a gamete; trisomies, in which three copies of a chromosome are present instead of the usual two, are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person’s body, or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells.
Chromosomal abnormalities that lead to disease in humans include
- Turner syndrome results from a single X chromosome (45, X or 45, X0).
- Klinefelter syndrome, the most common male chromosomal disease, otherwise known as 47, XXY is caused by an extra X chromosome.
- Edwards syndrome is caused by trisomy (three copies) of chromosome 18.
- Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21.
- Patau syndrome is caused by trisomy of chromosome 13.
- trisomy 8, trisomy 9 and trisomy 16, although they generally do not survive to birth.
Some disorders arise from loss of just a piece of one chromosome, including
- Cri du chat (cry of the cat), from a truncated short arm on chromosome 5. The name comes from the babies’ distinctive cry, caused by abnormal formation of the larynx.
- 1p36 Deletion syndrome, from the loss of part of the short arm of chromosome 1.
- Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the maternal genes, example of imprinting disorder
- Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the paternal genes, example of imprinting disorder.
Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual; one well-documented example is the Philadelphia chromosome, a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.