In a previous post, we have seen what is a chromosome, chromatid and chromatin. We know that the chromosome is condensed form of DNA, which is the genetic material of the cell. Chromosomes take up stain and appear as colourful bodies under microscope. The study of chromosomes is known as cytogenetics.
The chromosomes can be observed when properly extracted and stained. The proper X- shaped chromosomes are visible during the metaphase of the cell cycle. Staining the metaphase chromosomes allow them to be categorised according to their size, shape and characteristic banding patterns.
A karyotype is a technique wherein the chromosomes are stained, photographed and then each chromosome cutout is arranged according to their size and shape. This allows a glimpse at the entire genome of the cell. The abnormality at the entire or part of chromosome to bands level can be detected.
The preparation of karyotype needs the cells to divide, then be harvested and treated with certain chemicals, fixed, stained and observed. Let’s go through each step:
Theoretically any population of rapidly dividing cells can be used as the source of cell. This includes cells from tissue culture to cells from tumors. However the ideal cells are the peripheral blood lymphocytes, as they can be analyzed after only three days of culturing and the easily available (blood). Other cells which are used are skin fibroblasts and bone marrow cells. Chorionic villus cells and amniocytes are used specifically for genetic counseling. These cell types need around two weeks to divide to give sufficient amount of cells.
The mitogen triggers the cell to initiate cell division. This leads to the proliferation of the cells. E.g. Lymphocytes proliferate under the influence of mitogen like phytohemagglutinin (PHA).
– Metaphase arresting:
The karyotype needs metaphase chromosomes, most condensed form of chromosome. Hence the cells are arrested in the metaphase. This is done by treating the cultured cells with colcemid or colchicine, drugs which disrupts the mitotic spindle apparatus. Hence the cell cannot move to the next phase and most cells remain arrested in the metaphase.
– Hypotonic solution:
The cells arrested in the metaphase are then treated briefly with a hypotonic solution. In the hypotonic solution (less osmotic pressure) the cell swells and increase the visibility of the chromosomes. Usually 0.075M KCl is used.
– Slide preparation:
The cells are fixed using fixative. The Carnoy’s fixative is commonly used ((3:1 methanol: glacial acetic acid). Fixative kills the cells and also preserves the structure of cell and its components. These fixed cells are then dropped on a chilled microscope slide, wherein the swollen cells burst and the chromosomes spread. The chromosome spreads are then dried and stained. The most common stain used is the Giemsa stain. The dyes produce characteristic banding patterns. The chromosome spread are photographed using a camera attached to the microscope and the karyogram is prepared.
Chromosome spreads can be photographed, cut out, pasted with their homologous chromosomes and assigned to appropriate chromosome number and group. This can be done manually or they can be digitally imaged using a computer. There are softwares available to arrange the chromosomes.
In a normal human, there are seven groups (A-G) that 22 autosomal chromosomes are divided into based on size and position of the centromere (metacentric, submetacentric, acrocentric, and telocentric). There are two sex chromosomes, which decide the sex of the individual, they are X and Y. Females have two X chromosomes from each parent, males have one X from mother and a Y from father.
The chromosomes with their bands are then examined microscopically for abnormalities such as loss or gain of entire chromosomes, translocations of all or part of an arm of one chromosome to another, or more subtle changes in banding patterns associated with various genetic syndromes.
[Just for info: E.g. shown below is a karyotype of a female showing balanced translocation involving chromosome 9 and 15 (arrows) ie 46, XX, der (15), t(9q; 15q). Source: A newborn proband with der (15) and maternal karyotype 46,XX, der (15) t(9; 15) with a bad obstetric history. Panicker H et al, Vol. 53, No. 2, Journal of the Anatomical Society of India. Read full paper]
The standard nomenclature for describing a karyotype is based on the International System for Human Cytogenetic Nomenclature (ISCN). First, the total number of chromosomes are written followed by a comma, then the sex chromosome constitution and any abnormality written in parentheses. (46,XY:normal male)
• Advantage of karyotype
1. It is possible to analyze entire genome. It gives a lot of information before doing any in depth genetic analysis.
2. It is possible to visualize individual cells and individual chromosomes.
Genetic counsellors rely on karyotypes to diagnose abnormal pregnancies. Amniocentesis is a routine procedure used in prenatal screening that involves removing amniotic fluid for karyotype analysis.
A karyotype can detect polyploidy (specially in plants), aneuploidy, euploidy (i.e., Trisomy 21 or Down syndrome) and rearrangements such as deletions, duplications, and inversions.
Karyotyping allows spotting of a carrier, which may help predict the risk to future generations or pregnancies.
The karyotyping have a few limitations
– Resolution is limited to around 5Mb.
– Hence not all genetic abnormalities are detected by karyotype analysis. These include smaller abberations such as point mutations, frame shift mutations, nonsense mutations, or single nucleotide polymorphism’s.
– The karyotype can be done using only metaphase chromosomes and require actively growing cells. This cannot be carried on frozen or paraffin embedded samples.
– To have enough number of cells, the cells are need to be cultured, making the process time-consuming.
– The analysis requires skilled personnels for the analysis.
These disadvantages makes Fluorescence In Situ Hybridisation (FISH) the choice of technique. FISH can avoid the need to culture the cells and gives quicker results. Read about FISH posted by The Biotech Notes.
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Also read other posts by The Biotech Notes:
For Students preparing for CSIR-JRF-NET (Life Science, India), here are few books with good reviews.
Books on cytogenetics
Fundamentals of Cytogenetics and Genetics (by Mahabal Ram)
Cytogenetics (by P.K. Gupta)