Cytogenetics
Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology (a subdivision of human anatomy), that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis and meiosis.[1] Techniques used include karyotyping, analysis of G-banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).
History
Beginnings
Chromosomes were first observed in plant cells by Carl Nägeli in 1842. Their behavior in animal (salamander) cells was described by Walther Flemming, the discoverer of mitosis, in 1882. The name was coined by another German anatomist, von Waldeyer in 1888.
The next stage took place after the development of genetics in the early 20th century, when it was appreciated that the set of chromosomes (the
- Using cells in culture
- Pre-treating cells in a hypotonic solution, which swells them and spreads the chromosomes
- Arresting mitosis in metaphase by a solution of colchicine
- Squashing the preparation on the slide forcing the chromosomes into a single plane
- Cutting up a photomicrograph and arranging the result into an indisputable karyogram.
It took until 1956 for it to be generally accepted that the karyotype of man included only 46 chromosomes.
Applications of cytogenetics
McClintock's work on maize
Natural populations of Drosophila
In the 1930s,
Evidence rapidly accumulated to show that natural selection was responsible. Using a method invented by L'Héritier and Teissier, Dobzhansky bred populations in population cages, which enabled feeding, breeding and sampling whilst preventing escape. This had the benefit of eliminating migration as a possible explanation of the results. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. It was found that the various chromosome types do not fluctuate at random, as they would if selectively neutral, but adjust to certain frequencies at which they become stabilised. By the time Dobzhansky published the third edition of his book in 1951[16] he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes, as with most polymorphisms.[17][18]
Lily and mouse
The lily is a favored organism for the cytological examination of meiosis since the chromosomes are large and each morphological stage of meiosis can be easily identified microscopically. Hotta, Chandley et al.[19] presented the evidence for a common pattern of DNA nicking and repair synthesis in male meiotic cells of lilies and rodents during the zygotene–pachytene stages of meiosis when crossing over was presumed to occur. The presence of a common pattern between organisms as phylogenetically distant as lily and mouse led the authors to conclude that the organization for meiotic crossing-over in at least higher eukaryotes is probably universal in distribution.[citation needed]
Human abnormalities and medical applications
Following the advent of procedures that allowed easy enumeration of chromosomes, discoveries were quickly made related to aberrant chromosomes or chromosome number.[citation needed]
Constitutional cytogenetics: In some congenital disorders, such as Down syndrome, cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. In 1959, Lejeune[20] discovered patients with Down syndrome had an extra copy of chromosome 21. Down syndrome is also referred to as trisomy 21.
Other numerical abnormalities discovered include sex chromosome abnormalities. A female with only one X chromosome has Turner syndrome, whereas a male with an additional X chromosome, resulting in 47 total chromosomes, has Klinefelter syndrome. Many other sex chromosome combinations are compatible with live birth including XXX, XYY, and XXXX. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them, which is required in normal females to compensate for having two copies of the chromosome. Not all genes on the X chromosome are inactivated, which is why there is a phenotypic effect seen in individuals with extra X chromosomes.[citation needed]
Trisomy 13 was associated with
Acquired cytogenetics: In 1960, Peter Nowell and David Hungerford
Advent of banding techniques
In the late 1960s,
Diagrams identifying the chromosomes based on the banding patterns are known as idiograms. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid, and elongation techniques for all culture types that allow for higher-resolution banding.[citation needed]
Beginnings of molecular cytogenetics
In the 1980s, advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969, movement was now made in using fluorescent-labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescence in situ hybridization (FISH).[22] This change significantly increased the usage of probing techniques as fluorescent-labeled probes are safer. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated, cloned, and studied in ever greater detail.[citation needed]
Techniques
Karyotyping
The routine
Several chromosome-banding techniques are used in cytogenetics laboratories.
High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes (bands per haploid set, bph; "band level") increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.[23]
Slide preparation
Cells from
Analysis
Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patient's previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009)..
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations.
In addition to standard preparations FISH can also be performed on:
- bone marrow smears
- blood smears
- paraffin embedded tissue preparations
- enzymatically dissociated tissue samples
- uncultured bone marrow
- uncultured amniocytes
- Cytospinpreparations
Slide preparation
This section refers to the preparation of standard cytogenetic preparations
The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove the excess unbound probe, and counterstained with 4',6-Diamidino-2-phenylindole (DAPI) or propidium iodide.
Analysis
Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology, generally, a large number of interphase cells are scored in order to rule out low-level residual disease, generally between 200 and 1,000 cells are counted and scored. For congenital problems usually 20 metaphase cells are scored.[citation needed]
Future of cytogenetics
Advances now focus on
See also
References
- ISBN 978-0-387-07668-3
- ^ Levitsky, Grigorii Andreevich (1924). Material'nye osnovy nasledstvennosti [The Material Basis of Heredity] (in Russian). Kiev: Gosizdat Ukrainy.[page needed]
- ^ Levitsky GA (1931). "The morphology of chromosomes". Bull. Applied Bot. Genet. Plant Breed. 27: 19–174.
- ProQuest 1296285397.
- ^ von Winiwarter H (1912). "Études sur la spermatogenese humaine" [Human spermatogenesis studies]. Arch. Biologie (in French). 27 (93): 147–149.
- .
- .
- ^ Wright, Pearce (11 December 2001). "Joe Hin Tjio The man who cracked the chromosome count". The Guardian. Archived from the original on 25 August 2017.
- ^ Saxon, Wolfgang (7 December 2001). "Joe Hin Tjio, 82; Research Biologist Counted Chromosomes". The New York Times. Archived from the original on 12 May 2013.
- PMID 345813.
- ISBN 978-1-4612-6159-9.[page needed]
- ^ "Human genetics (Biology) :: The human chromosomes -- Britannica Online Encyclopedia". Archived from the original on 2011-02-17. Retrieved 2011-03-15. Encyclopædia Britannica, The Human Chromosome
- ^ "Chromosome fusion". Archived from the original on 2011-08-09. Retrieved 2010-05-29. Evolution Pages, Chromosome fusion
- PMID 23236127.
- PMID 17801695.
- ^ Dobzhansky T. 1951. Genetics and the origin of species. 3rd ed, Columbia University Press, New York.
- ^ Dobzhansky T. 1970. Genetics of the evolutionary process. Columbia University Press N.Y.
- ^ [Dobzhansky T.] 1981. Dobzhansky's genetics of natural populations. eds Lewontin RC, Moore JA, Provine WB and Wallace B. Columbia University Press N.Y.
- S2CID 4268089.
- NAID 10008406728.
- PMID 17739576.
- ISBN 978-81-7133-737-8.[page needed]
- PMID 23887773.
External links
- Cytogenetic Directory
- Cytogenetics Resources Archived 2017-05-26 at the Wayback Machine
- Human Cytogenetics - Chromosomes and Karyotypes
- Association for Genetic Technologists
- Association of Clinical Cytogeneticists
- Gladwin Medical Blog Archived 2006-11-08 at the Wayback Machine
- Cytogenetics - Technologies,markets and companies
- Cytogenetics-methods-and-trouble-shooting
- Department of Cytogenetics of Wikiversity