Cytogenetics is essentially a branch of
genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
, but is also a part of cell biology/cytology (a subdivision of human anatomy), that is concerned with how the
chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
s relate to cell behaviour, particularly to their behaviour during
mitosis
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is mainta ...
and
meiosis
Meiosis (; , since it is a reductional division) is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately resu ...
. Techniques used include
karyotyping
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
, analysis of
G-banded chromosomes, other cytogenetic banding techniques, as well as
molecular cytogenetics
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered tha ...
such as
fluorescent ''in situ'' hybridization (FISH) and
comparative genomic hybridization Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The ...
(CGH).
History
Beginnings
Chromosomes were first observed in plant cells by
Carl Nägeli
Carl Wilhelm von Nägeli (26 or 27 March 1817 – 10 May 1891) was a Swiss people, Swiss botany, botanist. He studied cell division and pollination but became known as the man who discouraged Gregor Mendel from further work on genetics. He reje ...
in 1842. Their behavior in animal (
salamander
Salamanders are a group of amphibians typically characterized by their lizard-like appearance, with slender bodies, blunt snouts, short limbs projecting at right angles to the body, and the presence of a tail in both larvae and adults. All ten ...
) cells was described by
Walther Flemming
Walther Flemming (21 April 1843 – 4 August 1905) was a German biologist and a founder of cytogenetics.
He was born in Sachsenberg (now part of Schwerin) as the fifth child and only son of the psychiatrist Carl Friedrich Flemming (1799–18 ...
, the discoverer of
mitosis
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is mainta ...
, 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
karyotype
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the
phenotypic
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
appearance of the
somatic
Somatic may refer to:
* Somatic (biology), referring to the cells of the body in contrast to the germ line cells
** Somatic cell, a non-gametic cell in a multicellular organism
* Somatic nervous system, the portion of the vertebrate nervous sys ...
chromosomes, in contrast to their
genic
''Genic'' (stylized as ''_genic'') is the twelfth and final studio album and final bilingual (English–Japanese) album by Japanese recording artist Namie Amuro. It was released on June 10, 2015 in three physical formats, and for digital consumpti ...
contents. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal
diploid
Ploidy () is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respectively ...
human cell contain? In 1912,
Hans von Winiwarter reported 47 chromosomes in
spermatogonia
A spermatogonium (plural: ''spermatogonia'') is an undifferentiated male germ cell. Spermatogonia undergo spermatogenesis to form mature spermatozoa in the seminiferous tubules of the testis.
There are three subtypes of spermatogonia in humans:
...
and 48 in
oogonia
An oogonium (plural oogonia) is a small diploid cell which, upon maturation, forms a primordial follicle in a female fetus or the female (haploid or diploid) gametangium of certain thallophytes.
In the mammalian fetus
Oogonia are formed in lar ...
, concluding an
XX/XO sex determination mechanism.
Painter
Painting is the practice of applying paint, pigment, color or other medium to a solid surface (called the "matrix" or "support"). The medium is commonly applied to the base with a brush, but other implements, such as knives, sponges, and ai ...
in 1922 was not certain whether the diploid number of humans was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and he correctly insisted on humans having an
XX/XY system of sex-determination. Considering their techniques, these results were quite remarkable. In science books, the number of human chromosomes remained at 48 for over thirty years. New techniques were needed to correct this error.
Joe Hin Tjio
Joe Hin Tjio (2 November 1919 – 27 November 2001), was an Indonesian-born American cytogeneticist. He was renowned as the first person to recognize the normal number of human chromosomes on December 22, 1955 at the Institute of Genetics of the ...
working in
Albert Levan
Albert Levan (8 March 1905 – 28 March 1998) was a Swedish botanist and geneticist.
Albert Levan is best known today for co-authoring the report in 1956 that humans had forty-six chromosomes (instead of forty-eight, as previously believed). Thi ...
's lab was responsible for finding the approach:
:# Using cells in culture
:# Pre-treating cells in a
hypotonic solution
In chemical biology, tonicity is a measure of the effective osmotic pressure gradient; the water potential of two solutions separated by a partially-permeable cell membrane. Tonicity depends on the relative concentration of selective membrane-i ...
, which swells them and spreads the chromosomes
:# Arresting
mitosis
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is mainta ...
in
metaphase
Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, align ...
by a solution of
colchicine
Colchicine is a medication used to treat gout and Behçet's disease. In gout, it is less preferred to NSAIDs or steroids. Other uses for colchicine include the management of pericarditis and familial Mediterranean fever. Colchicine is taken ...
:# 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. The
great apes
The Hominidae (), whose members are known as the great apes or hominids (), are a taxonomic family of primates that includes eight extant species in four genera: '' Pongo'' (the Bornean, Sumatran and Tapanuli orangutan); ''Gorilla'' (the east ...
have 48 chromosomes.
Human chromosome 2
Chromosome 2 is one of the twenty-three pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 2 is the second-largest human chromosome, spanning more than 242 million base pairs and representing almost ei ...
was formed by a merger of ancestral chromosomes, reducing the number.
Applications in cytogenetics
McClintock's work on maize
Barbara McClintock
Barbara McClintock (June 16, 1902 – September 2, 1992) was an American scientist and cytogeneticist who was awarded the 1983 Nobel Prize in Physiology or Medicine. McClintock received her PhD in botany from Cornell University in 1927. There s ...
began her career as a
maize
Maize ( ; ''Zea mays'' subsp. ''mays'', from es, maíz after tnq, mahiz), also known as corn (North American and Australian English), is a cereal grain first domesticated by indigenous peoples in southern Mexico about 10,000 years ago. Th ...
cytogeneticist. In 1931, McClintock and
Harriet Creighton
Harriet Baldwin Creighton (27 June 1909 – January 9, 2004) was an American botanist, geneticist and educator.
Background
Born in Delavan, Illinois, Creighton graduated from Wellesley College in 1929, and went on to complete her Ph.D. at Cor ...
demonstrated that cytological recombination of marked
chromosomes
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
correlated with recombination of genetic
traits (
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s). McClintock, while at the
Carnegie Institution
The Carnegie Institution of Washington (the organization's legal name), known also for public purposes as the Carnegie Institution for Science (CIS), is an organization in the United States established to fund and perform scientific research. T ...
, continued previous studies on the mechanisms of chromosome breakage and fusion flare in maize. She identified a particular chromosome breakage event that always occurred at the same locus on maize chromosome 9, which she named the "''Ds"'' or "dissociation" locus. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. During her cytogenetic work, McClintock discovered
transposon
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transpo ...
s, a find which eventually led to her
Nobel Prize
The Nobel Prizes ( ; sv, Nobelpriset ; no, Nobelprisen ) are five separate prizes that, according to Alfred Nobel's will of 1895, are awarded to "those who, during the preceding year, have conferred the greatest benefit to humankind." Alfr ...
in 1983.
Natural populations of Drosophila
In the 1930s,
Dobzhansky
Theodosius Grigorievich Dobzhansky (russian: Феодо́сий Григо́рьевич Добржа́нский; uk, Теодо́сій Григо́рович Добржа́нський; January 25, 1900 – December 18, 1975) was a prominent ...
and his coworkers collected ''
Drosophila pseudoobscura
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
'' and ''
D. persimilis'' from wild populations in
California
California is a U.S. state, state in the Western United States, located along the West Coast of the United States, Pacific Coast. With nearly 39.2million residents across a total area of approximately , it is the List of states and territori ...
and neighboring states. Using Painter's technique they studied the
polytene chromosomes and discovered that the wild populations were polymorphic for
chromosomal inversions
An inversion is a chromosome rearrangement in which a segment of a chromosome becomes inverted within its original position. An inversion occurs when a chromosome undergoes a two breaks within the chromosomal arm, and the segment between the two br ...
. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism.
Evidence rapidly accumulated to show that
natural selection
Natural selection is the differential survival and reproduction of individuals due to differences in phenotype. It is a key mechanism of evolution, the change in the heritable traits characteristic of a population over generations. Charle ...
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
Migration, migratory, or migrate may refer to: Human migration
* Human migration, physical movement by humans from one region to another
** International migration, when peoples cross state boundaries and stay in the host state for some minimum le ...
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 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes, as with most
polymorphisms.
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.
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.
Human abnormalities and medical applications
![T922 CML](https://upload.wikimedia.org/wikipedia/commons/2/2c/T922_CML.jpg)
Following the advent of procedures that allowed easy enumeration of chromosomes, discoveries were quickly made related to aberrant chromosomes or chromosome number. In some congenital disorders, such as
Down syndrome
Down syndrome or Down's syndrome, also known as trisomy 21, is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. It is usually associated with physical growth delays, mild to moderate intellectual dis ...
, cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. Abnormalities arising from
nondisjunction
Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate properly during cell division (mitosis/meiosis). There are three forms of nondisjunction: failure of a pair of homologous chromosomes to separate in meiosis I ...
events can cause cells with
aneuploidy
Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include a difference of one or more complete sets of chromosomes. A cell with any ...
(additions or deletions of entire chromosomes) in one of the parents or in the fetus. In 1959, Lejeune 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
Turner syndrome (TS), also known as 45,X, or 45,X0, is a genetic condition in which a female is partially or completely missing an X chromosome. Signs and symptoms vary among those affected. Often, a short and webbed neck, low-set ears, low hair ...
, whereas a male with an additional X chromosome, resulting in 47 total chromosomes, has
Klinefelter syndrome
Klinefelter syndrome (KS), also known as 47,XXY, is an aneuploid genetic condition where a male has an additional copy of the X chromosome. The primary features are infertility and small, poorly functioning testicles. Usually, symptoms are su ...
. 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.
Trisomy 13 was associated with
Patau syndrome
Patau syndrome is a syndrome caused by a chromosome, chromosomal abnormality, in which some or all of the Cell (biology), cells of the body contain Trisomy, extra genetic material from chromosome 13 (human), chromosome 13. The extra genetic mater ...
and trisomy 18 with
Edwards syndrome
Edwards syndrome, also known as trisomy 18, is a genetic disorder caused by the presence of a third copy of all or part of chromosome 18. Many parts of the body are affected. Babies are often born small and have heart defects. Other features in ...
.
In 1960, Peter Nowell and David Hungerford discovered a small chromosome in the white blood cells of patients with
Chronic myelogenous leukemia
Chronic myelogenous leukemia (CML), also known as chronic myeloid leukemia, is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulat ...
(CML). This abnormal chromosome was dubbed the
Philadelphia chromosome
The Philadelphia chromosome or Philadelphia translocation (Ph) is a specific genetic abnormality in chromosome 22 of leukemia cancer cells (particularly chronic myeloid leukemia (CML) cells). This chromosome is defective and unusually short becaus ...
- as both scientists were doing their research in
Philadelphia, Pennsylvania
Philadelphia, often called Philly, is the largest city in the Commonwealth of Pennsylvania, the sixth-largest city in the U.S., the second-largest city in both the Northeast megalopolis and Mid-Atlantic regions after New York City. Sinc ...
. Thirteen years later, with the development of more advanced techniques, the abnormal chromosome was shown by
Janet Rowley
Janet Davison Rowley (April 5, 1925 – December 17, 2013) was an American human geneticist and the first scientist to identify a chromosomal translocation as the cause of leukemia and other cancers, thus proving that cancer is a genetic disease. ...
to be the result of a
translocation of chromosomes 9 and 22. Identification of the Philadelphia chromosome by cytogenetics is diagnostic for CML.
Advent of banding techniques
![Human karyotype with bands and sub-bands](https://upload.wikimedia.org/wikipedia/commons/b/b1/Human_karyotype_with_bands_and_sub-bands.png)
In the late 1960s,
Torbjörn Caspersson
Torbjörn Oskar Caspersson (15 October 1910 – 7 December 1997) was a Swedish cytologist and geneticist. He was born in Motala and attended the University of Stockholm, where he studied medicine and biophysics.
Contributions
Caspersson made sev ...
developed a quinacrine fluorescent staining technique (Q-banding) which revealed unique banding patterns for each chromosome pair. This allowed chromosome pairs of otherwise equal size to be differentiated by distinct horizontal banding patterns. Banding patterns are now used to elucidate the breakpoints and constituent chromosomes involved in
chromosome translocations. Deletions and inversions within an individual chromosome can also be identified and described more precisely using standardized banding nomenclature. G-banding (utilizing trypsin and Giemsa/ Wright stain) was concurrently developed in the early 1970s and allows visualization of banding patterns using a bright field microscope.
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
The amniotic fluid is the protective liquid contained by the amniotic sac of a gravid amniote. This fluid serves as a cushion for the growing fetus, but also serves to facilitate the exchange of nutrients, water, and biochemical products betwee ...
, and elongation techniques for all culture types that allow for higher-resolution banding.
Beginnings of molecular cytogenetics
In the 1980s, advances were made in
molecular cytogenetics
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered tha ...
. 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). 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.
Techniques
Karyotyping
The routine chromosome analysis (
Karyotyping
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
) refers to analysis of
metaphase
Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, align ...
chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
s which have been banded using
trypsin
Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the dig ...
followed by
Giemsa
Giemsa stain (), named after German chemist and bacteriologist Gustav Giemsa, is a nucleic acid stain used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.
Uses
It is specific for the phosphate groups of ...
, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns are unknown, although it likely related to
replication timing
Replication timing refers to the order in which segments of DNA along the length of a chromosome are duplicated.
DNA replication
In eukaryotic cells (cells that package their DNA within a nucleus), chromosomes consist of very long linear doubl ...
and chromatin packing.
Several chromosome-banding techniques are used in cytogenetics laboratories.
Quinacrine
Mepacrine, also called quinacrine or by the trade name Atabrine, is a medication with several uses. It is related to chloroquine and mefloquine. Although formerly available from compounding pharmacies, as of August 2020 it is unavailable in th ...
banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as
Giemsa
Giemsa stain (), named after German chemist and bacteriologist Gustav Giemsa, is a nucleic acid stain used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.
Uses
It is specific for the phosphate groups of ...
banding (G-banding). Reverse banding, or R-banding, requires heat treatment and reverses the usual black-and-white pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and
nucleolar
The nucleolus (, plural: nucleoli ) is the largest structure in the nucleus of eukaryotic cells. It is best known as the site of ribosome biogenesis, which is the synthesis of ribosomes. The nucleolus also participates in the formation of s ...
organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the
constitutive heterochromatin
Constitutive heterochromatin domains are regions of DNA found throughout the chromosomes of eukaryotes. The majority of constitutive heterochromatin is found at the pericentromeric regions of chromosomes, but is also found at the telomeres and thro ...
, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of
acrocentric chromosomes.
High-resolution banding involves the staining of chromosomes during
prophase
Prophase () is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin ret ...
or early
metaphase
Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, align ...
(prometaphase), before they reach maximal condensation. Because
prophase
Prophase () is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin ret ...
and
prometaphase
Prometaphase is the phase of mitosis following prophase and preceding metaphase, in eukaryotic somatic cells. In prometaphase, the nuclear membrane breaks apart into numerous "membrane vesicles", and the chromosomes inside form protein struc ...
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.
Slide preparation
Cells from
bone marrow
Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...
, blood, amniotic fluid,
cord blood
Cord blood (umbilical cord blood) is blood that remains in the placenta and in the attached umbilical cord after childbirth. Cord blood is collected because it contains stem cells, which can be used to treat hematopoietic and genetic disorders s ...
, tumor, and tissues (including skin,
umbilical cord
In placental mammals, the umbilical cord (also called the navel string, birth cord or ''funiculus umbilicalis'') is a conduit between the developing embryo or fetus and the placenta. During prenatal development, the umbilical cord is physiologic ...
, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A
mitotic inhibitor
A mitotic inhibitor is a drug that inhibits mitosis, or cell division. These drugs disrupt microtubules, which are structures that pull the chromosomes apart when a cell divides. Mitotic inhibitors are used in cancer treatment, because cancer cell ...
(
colchicine
Colchicine is a medication used to treat gout and Behçet's disease. In gout, it is less preferred to NSAIDs or steroids. Other uses for colchicine include the management of pericarditis and familial Mediterranean fever. Colchicine is taken ...
,
colcemid) is then added to the culture. This stops cell division at
mitosis
In cell biology, mitosis () is a part of the cell cycle in which replicated chromosomes are separated into two new nuclei. Cell division by mitosis gives rise to genetically identical cells in which the total number of chromosomes is mainta ...
which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic solution, Carnoy's fixative (3:1
methanol
Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
to
glacial acetic acid
Acetic acid , systematically named ethanoic acid , is an acidic, colourless liquid and organic compound with the chemical formula (also written as , , or ). Vinegar is at least 4% acetic acid by volume, making acetic acid the main component ...
) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis.
Analysis
Analysis of banded chromosomes is done at a
microscope
A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisibl ...
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)..
Fluorescent in situ hybridization
Fluorescent in situ hybridization
Fluorescence ''in situ'' hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed ...
(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
*
Cytospin preparations
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
Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an Alcohol (chemistry), alcohol with the chemical formula . Its formula can be also written as or (an ethyl ...
, 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
DAPI (pronounced 'DAPPY', /ˈdæpiː/), or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an inta ...
) or propidium iodide.
Analysis
Analysis of FISH specimens is done by
fluorescence microscopy
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscop ...
by a clinical laboratory specialist in cytogenetics. For oncology, generally, a large number of
interphase
Interphase is the portion of the cell cycle that is not accompanied by visible changes under the microscope, and includes the G1, S and G2 phases. During interphase, the cell grows (G1), replicates its DNA (S) and prepares for mitosis (G2). A c ...
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.
Future of cytogenetics
Advances now focus on
molecular cytogenetics
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered tha ...
including automated systems for counting the results of standard FISH preparations and techniques for
virtual karyotyping, such as comparative genomic hybridization arrays, CGH and
Single nucleotide polymorphism
In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
arrays.
See also
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Cytotaxonomy
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Karyotype
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
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Molecular cytogenetics
Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered tha ...
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Ploidy
Ploidy () is the number of complete sets of chromosomes in a cell (biology), cell, and hence the number of possible alleles for Autosome, autosomal and Pseudoautosomal region, pseudoautosomal genes. Sets of chromosomes refer to the number of mat ...
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Virtual karyotype
Virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated. It detects genomic copy number variations at a hig ...
References
External links
Cytogenetic DirectoryHuman Cytogenetics - Chromosomes and KaryotypesAssociation for Genetic TechnologistsAssociation of Clinical CytogeneticistsCytogenetics-methods-and-trouble-shooting*
Department of Cytogenetics of Wikiversity
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