The chromosome analysis involves many conditions such as congenital malformations, mental retardation, repeated abortions, sex determination, and prenatal diagnosis. The procedure of chromosome analysis involves techniques such as karyotyping and FISH. A microscopic examination of chromosomes for detection of abnormalities or novel mutations involves chromosome analysis. It helps to detect monosomy, trisomy, infertility issues, repeated abortions, hereditary syndromes, and chromosomal aberrations. It helps in detecting the disorder or a disease. It helps in the genetic counseling and consultation. It is possible to diagnose hematological disorders as well. Modern FISH techniques detect cytogenetic abnormalities in haemato-lymphoid malignancies and hereditary cancers. Let us know the technique in detail.
Karyotyping:
It is a procedure to obtain karyotype of an individual. A karyotype is nothing but a chromosome complement of an individual. It depicts a photomicrograph of the metaphase chromosome arranged in a standard sequence. The review article describes the procedure in detail.
The human peripheral blood lymphocytes culturing and processing helps to obtain a karyotype. A sterile syringe is used to collect the sample. It involves the collection of 5ml of the nervous blood in a heparinized tube. Addition of heparin prevents blood clotting. The procedure primarily involves the addition of the heparinized whole blood sample into the culture medium consisting of RPMI-1640, fetal calf serum, phytohaemagglutinin, and antibiotics. The culture media and the fetal calf serum nourish the lymphocytes. Phytohaemagglutinin stimulates cell division in lymphocytes. Addition of the antibiotics prevents the infection. The culture vial involves incubation of 3 days at 370C temperature.
The incubation of the culture vials leads to the division of the lymphocytic cells. The addition of a mitotic inhibitor (colchicine) at the end of the third day, helps a lot by preventing the spindles. It arrests the cells in metaphase. Visibility of the chromosomes is best during the metaphase. Then the culture vials are kept undisturbed for two hours. After 2 hours the lymphocytes are centrifuged. Then the cells undergo saline treatment. The saline creates a hypotonic treatment. The cells swell under the hypotonic environment. Followed by re-centrifugation, the procedure requires removal of the supernatant. After discarding the supernatant, the cells get fixed in a fixative such as Carnoy’s fixative. A careful dropping of few drops of the cell suspension on a clean, pre-chilled, grease free slide enables the chromosomes to get dispersed. The processing of the slides and staining them helps to visualize the chromosomes under a microscope. Karyotyping software helps to arrange the chromosomes in groups thereby generating a photomicrograph of chromosomes known as a karyotype.
Observation of the chromosomes:
A karyotype reflects the differences in the absolute sizes of the chromosomes. It helps to study the DNA duplication. The differences in the relative sizes of the chromosomes arise due to the interchange of the chromosomal segments. The chromosomal segments may interchange in unequal lengths. A karyotype helps to observe various types of chromosomal abnormalities known as deletions, duplications, translocations, and inversions. For example, a translocation arises due to differences in the Centromeric position. Grouping of the chromosomes helps in identifying the correct number of the chromosomes. Also, the karyotyping enables to study the number and the position of the satellites.
It commonly helps in analyzing the chromosomes. Giemsa banding technique first involves treatment of the chromosomes with a denaturing agent known as trypsin. It helps in denaturing the proteins. A stain known as Giemsa helps to stain the slides. Staining with the Giemsa stain creates a unique banding pattern to the chromosomes. The pattern shows light and dark bands. A specific banding pattern helps to visualize the long and short arms of the chromosome. Giemsa stain is a complex stain specific to the phosphate groups of the DNA. The light and dark stripes appearing on the chromosomal arms depict the bands. They appear after staining the cell preparation.
The heterochromatin depicts a dark band. The euchromatin depicts a light band. Heterochromatin is rich in the repetitive DNA sequences. Other types of banding techniques include R-banding, C-banding, Q-banding, T-banding and silver staining.
Three main types of karyotyping include classical karyotyping, spectral karyotyping, and virtual karyotyping. The above method of karyotyping involves Giemsa banding belonging to the classical karyotyping. The spectral karyotyping helps to visualize all the chromosomes in different colors. This type of karyotyping is known as SKY technique. A digital karyotype helps to quantify the DNA copy number.
Image : Giemsa banding of chromosomes
Fluorescence in situ hybridization (FISH):
A metaphase chromosome consists of a DNA packed into it in a highly condensed form. The DNA sequence helps to detect the defects. The FISH technique uses a single-stranded DNA probe. It has a unique ability to anneal with the complementary DNA sequence on the chromosome. A DNA probe is a single-stranded sequence. It gets labeled radioactively. The DNA probe detects the DNA fragments with similar sequences. The hybridization of the DNA sequence with the probe sequence enables it to be visualized using autoradiography.
The FISH technique involves different types of probes. The Centromeric probes are nothing but the DNA sequences found in and around the centromere. They are specific to a particular chromosome and have a repetitive sequence. A chromosome-specific unique sequence probe identifies sub-microscopic deletions and duplications. A whole chromosome paint helps to visualize an entire chromosome. Examination of slides under a microscope detects the presence of a hybridized fluorescent signal. It also detects the absence of the chromosomal material if there is no signal. Not only metaphase chromosomes but also non-metaphase chromosomes such as interphase chromosomes hybridize with the fluorescent labeled probes.
Chromosome painting is the next version of FISH. In this technique, the hybridization probe involves a mixture of DNA molecules specific for different regions of a single chromosome. References:
[1] Medical genetics, G.P. Pal
[2] Molecular Cytogenetics: Protocols and Applications, Yao-Shan Fan
[3] Human Chromosomes: Structure, Behavior, and Effects, Eeva Therman, Millard Susman
[4] The AGT Cytogenetics Laboratory Manual, Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce
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