Cytogenetics
Encyclopedia
Cytogenetics is a branch of genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
that is concerned with the study of the structure and function of the cell, especially the chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s. It includes routine analysis of G-Banded
G banding
G-banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes.It is useful for identifying various genetic diseases through the photographic representation of the entire chromosome complement. The metaphase chromosomes are treated with trypsin and...
chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics
Molecular cytogenetics
Molecular cytogenetics involves the combination of molecular biology and cytogenetics. In general this involves the use of a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or...
such as fluorescent in situ hybridization
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
(FISH) and comparative genomic hybridization
Comparative genomic hybridization
Comparative genomic hybridization or Chromosomal Microarray Analysis is a molecular-cytogenetic method for the analysis of copy number changes in the DNA content of a given subject's DNA and often in tumor cells....
(CGH).
Early years
Chromosomes were first observed in plant cells by Karl Wilhelm von NägeliKarl Wilhelm von Nägeli
Karl Wilhelm von Nägeli was a Swiss botanist. He studied cell division and pollination, but became known as the man who discouraged Gregor Mendel from further work on genetics.-Birth and education:...
in 1842. Their behavior in animal (salamander
Salamander
Salamander is a common name of approximately 500 species of amphibians. They are typically characterized by a superficially lizard-like appearance, with their slender bodies, short noses, and long tails. All known fossils and extinct species fall under the order Caudata, while sometimes the extant...
) cells was described by Walther Flemming
Walther Flemming
Walther Flemming was a German biologist and a founder of cytogenetics.He was born in Sachsenberg near Schwerin as the fifth child and only son of the psychiatrist Carl Friedrich Flemming and his second wife, Auguste Winter...
, the discoverer of mitosis
Mitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
, in 1882. The name was coined by another German anatomist, von Waldeyer
Heinrich Wilhelm Gottfried von Waldeyer-Hartz
Heinrich Wilhelm Gottfried von Waldeyer-Hartz was a German anatomist, famous for consolidating the neuron theory of organization of the nervous system and for naming the chromosome...
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
Karyotype
A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.p28...
) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic
Somatic
The term somatic means 'of the body',, relating to the body. In medicine, somatic illness is bodily, not mental, illness. The term is often used in biology to refer to the cells of the body in contrast to the germ line cells which usually give rise to the gametes...
chromosomes, in contrast to their genic
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
contents. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912, Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia, concluding an XX/XO sex determination
Sex-determination system
A sex-determination system is a biological system that determines the development of sexual characteristics in an organism. Most sexual organisms have two sexes. In many cases, sex determination is genetic: males and females have different alleles or even different genes that specify their sexual...
mechanism. Painter
Theophilus Painter
Theophilus Shickel Painter was an American zoologist known for his work in identifying genes in fruit flies...
in 1922 was not certain whether the diploid number of man was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and he correctly insisted on man having an XX/XY
XY sex-determination system
The XY sex-determination system is the sex-determination system found in humans, most other mammals, some insects and some plants . In this system, females have two of the same kind of sex chromosome , and are called the homogametic sex. Males have two distinct sex chromosomes , and are called...
system. Considering their techniques, these results were quite remarkable.
New techniques were needed to definitively solve the problem:
-
- Using cells in culture
- Pre-treating cells in a hypotonic solution, which swells them and spreads the chromosomes
- Arresting mitosisMitosisMitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
in metaphaseMetaphaseMetaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
by a solution of colchicineColchicineColchicine is a medication used for gout. It is a toxic natural product and secondary metabolite, originally extracted from plants of the genus Colchicum... - 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 until it became generally accepted that the karyotype of man included only 46 chromosomes. Rather interestingly, the great apes
Great Apes
Great Apes may refer to*Great apes, species in the biological family Hominidae, including humans, chimpanzees, gorillas, and orangutans*Great Apes , a 1997 novel by Will Self...
have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes, reducing the number.
McClintock's work on maize
Barbara McClintockBarbara McClintock
Barbara McClintock , the 1983 Nobel Laureate in Physiology or Medicine, was an American scientist and one of the world's most distinguished cytogeneticists. McClintock received her PhD in botany from Cornell University in 1927, where she was a leader in the development of maize cytogenetics...
began her career as a maize
Maize
Maize known in many English-speaking countries as corn or mielie/mealie, is a grain domesticated by indigenous peoples in Mesoamerica in prehistoric times. The leafy stalk produces ears which contain seeds called kernels. Though technically a grain, maize kernels are used in cooking as a vegetable...
cytogeneticist. In 1931, McClintock and Harriet Creighton
Harriet Creighton
Harriet Baldwin Creighton was an American botanist, geneticist and educator.Born in Delavan, Illinois, Creighton graduated from Wellesley College in 1929, and went on to complete her Ph.D. at Cornell University in 1933...
demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s). 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
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s, a find which eventually led to her Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
in 1983.
Natural populations of Drosophila
In the 1930s, Dobzhansky and his co-workers collected Drosophila pseudoobscuraDrosophila pseudoobscura
Drosophila pseudoobscura is a species of fruit fly, used extensively in lab studies of speciation.In 2005, D. pseudoobscura was the second Drosophila species to have its genome sequenced, after the model organism Drosophila melanogaster....
and D. persimilis from wild populations in California
California
California is a state located on the West Coast of the United States. It is by far the most populous U.S. state, and the third-largest by land area...
and neighboring states. Using Painter's technique they studied the polytene chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. 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
Natural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
was responsible. Using a method invented by L'Heretier 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 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes, as with most polymorphisms
Polymorphism (biology)
Polymorphism in biology occurs when two or more clearly different phenotypes exist in the same population of a species — in other words, the occurrence of more than one form or morph...
.
Human abnormalities and medical applications
Nondisjunction
Nondisjunction is the failure of chromosome pairs to separate properly during meiosis stage 1 or stage 2. This could arise from a failure of homologous chromosomes to separate in meiosis I, or the failure of sister chromatids to separate during meiosis II or mitosis. The result of this error is a...
events can cause cells with aneuploidy
Aneuploidy
Aneuploidy is an abnormal number of chromosomes, and is a type of chromosome abnormality. An extra or missing chromosome is a common cause of genetic disorders . Some cancer cells also have abnormal numbers of chromosomes. Aneuploidy occurs during cell division when the chromosomes do not separate...
(additions or deletions of entire chromosomes) in one of the parents or in the fetus. In 1959, Lejeune discovered patients with Down syndrome
Down syndrome
Down syndrome, or Down's syndrome, trisomy 21, is a chromosomal condition caused by the presence of all or part of an extra 21st chromosome. It is named after John Langdon Down, the British physician who described the syndrome in 1866. The condition was clinically described earlier in the 19th...
had an extra copy of chromosome 21. Down syndrome is also referred to as trisomy 21.
Other numerical abnormalities discovered include sex chromosome abnormalities. An individual with only one sex chromosome (the X) has Turner syndrome
Turner syndrome
Turner syndrome or Ullrich-Turner syndrome encompasses several conditions in human females, of which monosomy X is most common. It is a chromosomal abnormality in which all or part of one of the sex chromosomes is absent...
, an additional X chromosome in a male, resulting in 47 total chromosomes, has Klinefelter's Syndrome
Klinefelter's syndrome
Klinefelter syndrome, 46/47, XXY, or XXY syndrome is a condition in which human males have an extra X chromosome. While females have an XX chromosomal makeup, and males an XY, affected individuals have at least two X chromosomes and at least one Y chromosome...
. 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
Barr body
A Barr body is the inactive X chromosome in a female somatic cell, rendered inactive in a process called lyonization, in those species in which sex is determined by the presence of the Y or W chromosome rather than the diploidy of the X or Z...
, 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's Syndrome and trisomy 18 with Edward's Syndrome.
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
Chronic myelogenous leukemia , also known as chronic granulocytic leukemia , is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood...
(CML). This abnormal chromosome was dubbed the Philadelphia chromosome
Philadelphia chromosome
Philadelphia chromosome or Philadelphia translocation is a specific chromosomal abnormality that is associated with chronic myelogenous leukemia . It is the result of a reciprocal translocation between chromosome 9 and 22, and is specifically designated t...
- as both scientists were doing their research in Philadelphia, Pennsylvania
Philadelphia, Pennsylvania
Philadelphia is the largest city in the Commonwealth of Pennsylvania and the county seat of Philadelphia County, with which it is coterminous. The city is located in the Northeastern United States along the Delaware and Schuylkill rivers. It is the fifth-most-populous city in the United States,...
. Thirteen years later, with the development of more advanced techniques, the abnormal chromosome was shown by Janet Rowley
Janet Rowley
Janet Davison Rowley is an American human geneticist and the first scientist to identify a chromosomal translocation as the cause of leukemia and other cancers....
to be the result of a translocation
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
of chromosomes 9 and 22. Identification of the Philadelphia chromosome by cytogenetics, in addition to other tests, is used today as a diagnostic for CML.
Advent of banding techniques
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
. Deletions within one chromosome could also now be more specifically named and understood. Deletion syndromes such as DiGeorge syndrome
DiGeorge syndrome
22q11.2 deletion syndrome, which has several presentations including DiGeorge syndrome , DiGeorge anomaly, velo-cardio-facial syndrome, Shprintzen syndrome, conotruncal anomaly face syndrome, Strong syndrome, congenital thymic aplasia, and thymic hypoplasia is a syndrome caused by the deletion of a...
, Prader-Willi syndrome
Prader-Willi syndrome
Prader–Willi syndrome is a rare genetic disorder in which seven genes on chromosome 15 are deleted or unexpressed on the paternal chromosome...
and others were discovered to be caused by deletions in chromosome material.
Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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.
Beginnings of molecular cytogenetics
In the 1980s, advances were made in molecular cytogeneticsMolecular cytogenetics
Molecular cytogenetics involves the combination of molecular biology and cytogenetics. In general this involves the use of a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or...
. While radioisotope-labeled probes had been hybridized with DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
since 1969, movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
(FISH). This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection
Chromosome microdissection
Chromosome microdissection is a technique that physically removes a large section of DNA from a complete chromosome. The smallest portion of DNA that can be isolated using this method comprises 10 million base pairs - hundreds or thousands of individual genes....
whereby aberrations in chromosomal structure could be isolated, cloned and studied in ever greater detail.
Karyotyping
Routine chromosome analysis (Karyotyping) refers to analysis of metaphaseMetaphase
Metaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s which have been banded using trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing
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 , chromosomes consist of very long linear double-stranded DNA molecules...
and chromatin packing.
Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine
Quinacrine
Quinacrine is a drug with a number of different medical applications. It is related to mefloquine.-Uses:Its main effects are as an antiprotozoal, antirheumatic and an intrapleural sclerosing agent....
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 banding (G-banding). Reverse banding (R-banding) requires heat treatment and reverses the usual white and black 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 organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin
Heterochromatin
Heterochromatin is a tightly packed form of DNA, which comes in different varieties. These varieties lie on a continuum between the two extremes of constitutive and facultative heterochromatin...
, 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
Prophase, from the ancient Greek πρό and φάσις , is a stage of mitosis in which the chromatin condenses into a highly ordered structure called a chromosome in which the chromatin becomes visible. This process, called chromatin condensation, is mediated by the condensin complex...
or early metaphase
Metaphase
Metaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
(prometaphase), before they reach maximal condensation. Because prophase
Prophase
Prophase, from the ancient Greek πρό and φάσις , is a stage of mitosis in which the chromatin condenses into a highly ordered structure called a chromosome in which the chromatin becomes visible. This process, called chromatin condensation, is mediated by the condensin complex...
and prometaphase
Prometaphase
Prometaphase is the phase of mitosis following prophase and preceding metaphase, in eukaryotic somatic cells. In Prometaphase, The nuclear envelope breaks into fragments and disappears. The tiny nucleolus inside the nuclear envolope, also dissolves. Microtubules emerging from the centrosomes at the...
chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes 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, blood, amniotic fluid, cord bloodCord 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.-Collection:...
, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor
Mitotic inhibitor
A mitotic inhibitor is a drug that inhibits mitosis, or cell division. These drugs disrupt microtubules, which are structures that pull the cell apart when it divides...
(colchicine, colcemid) is then added to the culture. This stops cell division at mitosis
Mitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
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, Carnoy's fixative (3:1 methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
to glacial acetic acid
Acetic acid
Acetic acid is an organic compound with the chemical formula CH3CO2H . It is a colourless liquid that when undiluted is also called glacial acetic acid. Acetic acid is the main component of vinegar , and has a distinctive sour taste and pungent smell...
) 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 microscopeMicroscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
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 patients 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 hybridizationFluorescent in situ hybridizationFISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
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 smearsBlood filmA blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examined microscopically...
- paraffin embedded tissue preparations
- enzymatically dissociated tissue samples
- uncultured bone marrow
- uncultured amniocytesAmniocyteAn amniocyte is a cell of a fetus which is suspended in the amniotic fluid....
- cytospin preparations
Slide preparation
This section refers to preparation of standard cytogenetic preparationsThe slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
, and the probe mixture is added. The sample DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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 excess unbound probe, and counterstained with 4',6-Diamidino-2-phenylindole (DAPI
DAPI
DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy...
) or propidium iodide.
Analysis
Analysis of FISH specimens is done by fluorescence microscopyFluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase
Interphase
Interphase is the phase of the cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for cell division. In preparation for cell division, it increases its size and makes a copy of its DNA...
cells are scored in order to rule out low level residual disease, generally between 200 and 1000 cells are counted and scored. For congenital problems usually 20 metaphase cells are scored.
Future of cytogenetics
Advances now focus on molecular cytogeneticsMolecular cytogenetics
Molecular cytogenetics involves the combination of molecular biology and cytogenetics. In general this involves the use of a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or...
including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping
Virtual Karyotype
Virtual karyotype detects genomic copy number variations at a higher resolution level than conventional karyotyping or chromosome-based comparative genomic hybridization .-Background:...
, such as comparative genomic hybridization arrays, CGH and Single nucleotide polymorphism
Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
-arrays.