A metaphase cell positive for the bcr/abl rearrangement using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (up left) is the one where the wrong rearrangement is present

FISH (Fluorescent in situ hybridization) is a cytogenetic technique which can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. FISH is often used for finding specific features in DNA. These features can be used in genetic counseling, medicine, and species identification. FISH image of bcr/abl positive rearranged metaphase File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... FISH image of bcr/abl positive rearranged metaphase File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... In situ is a Latin phrase meaning in the place. ... Hybridisation is the process of combining complementary, single-stranded nucleic acids into a single molecule. ... A metaphase cell positive for the bcr/abl rearrangement using FISH Cytogenetics is the study of the structure of chromosome material. ... The structure of part of a DNA double helix Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living organisms. ... part of a DNA sequence A DNA sequence (sometimes genetic sequence) is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand (adenine, cytosine, guanine... Figure 1: A representation of a condensed eukaryotic chromosome, as seen during cell division. ... A hybridization probe is a short piece of DNA (on the order of 100-500 bases) that is denatured (by heating) into single strands and then radioactively labeled, usually with phosphorus (32P or 33P). ... Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye. ...

Probes

Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use in the Human Genome Project. The size of the human genome is so large compared to the length that could be sequenced directly that it was necessary to divide the genome into fragments. The fragments were added into a framework that made it possible to use bacteria to replicate the fragments. The fragments were put into order by analyzing size-exclusion separation of enzymatically-digested fragments. Clonal populations of bacteria, each population maintaining a single artificial chromosome, are stored in various laboratories around the world. The artificial chromosomes (BAC) can be grown, extracted, and labeled, in any lab. These fragments are on the order of 100 thousand base-pairs, and are the basis for most FISH probes. A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. ...

Preparation and Hybridization Process

First, a probe is constructed. The probe has to be long enough to hybridize specifically to its target (and not to similar sequences in the genome), but not too large to impede the hybridization process, and it should be tagged directly with fluorophores, with targets for antibodies or with biotin. This can be done in various ways, for example nick translation and PCR using tagged nucleotides. Image File history File links No higher resolution available. ... In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. ... A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ... Wikipedia does not yet have an article with this exact name. ... Vitamin H redirects here. ... Nick translation is a tagging technique from molecular biology in which endonucleases are used to remove some of the nucleotides of a DNA sequence. ... Wikipedia does not yet have an article with this exact name. ... A nucleotide is a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups. ...

Then, an interphase or metaphase chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample. The probe is then applied to the chromosome DNA and incubated for ~12 hours while hybridizing. Several wash steps remove all unhybridized or partially hybridized probes. The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images. This article or section does not cite its references or sources. ... An image of a newt lung cell stained with flourescent dyes during metaphase. ... In biochemistry, a substrate is a molecule upon which an enzyme acts. ...

If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. The signal strength depends on many factors; probe labeling efficiency, the type of probe and the type of dye effect the fluorescent signal. Fluorescently-tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal. Robert Hookes microscope (1665) - an engineered device used to study living systems. ... Wikipedia does not yet have an article with this exact name. ... Streptavidin (less commonly spelled streptavidine) is a tetrameric protein purified from Streptomyces avidinii that binds very tightly to the vitamin biotin with a dissociation constant (Kd) of ~ 10^(–15) M. This is one of the strongest biochemical interactions known, and is widely taken advantage of in scientific laboratories. ...

Fibre FISH

An alternative to interphase or metaphase preparations, fibre FISH, interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line, rather than being tightly coiled, as in conventional FISH, or adopting a random conformation, as in interphase FISH. This is accomplished by applying mechanical shear along the length of the slide; either to cells which have been fixed to the slide and then lysed, or to a solution of purified DNA. A technique known as chromosome combing is increasingly used for this purpose. The extended conformation of the chromosomes allows dramatically higher resolution - even down to a few kilobases. The preparation of fibre FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely. This article or section does not cite its references or sources. ... In physics and mechanics, shear refers to a deformation that causes parallel surfaces to slide past one another (as opposed to compression and tension, which cause parallel surfaces to move towards or away from one another). ... Lysis (Greek lusis from luein = to separate) is the reduction of symptoms of a disease the dissolving of cells osmotic lysis chemical lysis viral lysis a dialogue of Plato about friendship (philia) This is a disambiguation page — a navigational aid which lists other pages that might otherwise share the... Chromosome Combing (also known as molecular combing or DNA combing) is a technique used to produce an array of uniformly stretched DNA that is then highly suitable for hybridisation studies such as FISH (fluorescent in situ hybridization) which benefit from the uniformity of stretching, the easy access to the hybridisation... A unit of measurement in molecular biology denoting a length of 1000 nucleotides (bases) of DNA or RNA. Categories: | ...

Variations on Probes and Analysis

FISH is a very general technique. It is often arbitrarily divided into more specific categories based on application, however each category is similar in that, in a chemical sense, the technique is the same; hybridization is the common denominator. The differences between the various FISH techniques are usually due to the construction and content of the fluorescently-labeled DNA probe. The size, overlap, colour, and mixture of the probes make possible all FISH techniques.

Interphase cells positive for a chromosomal (t9;22) rearrangement.

Probes size is important because longer probes hybridize more specifically than shorter probes. The overlap defines the resolution of detectable features. If the goal of an experiment is to detect the break point of a translocation, then the overlap of the probes—the degree to which one DNA sequence is contained in the adjacent probes—defines the minimum window in which the breakpoint occurs. fish bcr/abl cells File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... fish bcr/abl cells File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Chromosomal translocation of the 4th and 20th chromosome. ...

The mixture of probes determines the type of feature the probe can detect. Probes that hybridize along an entire chromosome are used to count the number of a certain chromosome, show translocations, or identify extra-chromosomal fragments of chromatin. This is often called "whole-chromosome painting." If every possible probe is used, every chromosome, (in essence the whole genome) would be marked fluorescently, which would not be particularly useful for determining features of individual sequences. A mixture of smaller probes can be created that are specific to a particular region (locus) of DNA; these mixtures are used to detect deletion mutations. When combined with a specific colour, a locus-specific probe mixture is used to detect very specific translocations. Special locus-specific probe mixtures are often used to count chromosomes, by binding to the centromeric regions of chromosomes, which are unique enough to identify each chromosome (with the exception of Chromosome 13, 14 21, 22.) Chromatin is the complex of DNA and protein found inside the nuclei of eukaryotic cells. ... A genetic deletion is a genetic aberration in which part of a chromosome is missing. ... The word locus (plural loci) is Latin for place: In biology and evolutionary computation, a locus is the position of a gene (or other significant sequence) on a chromosome. ... There are very few or no other articles that link to this one. ... Chromosome 13 is one of the 23 pairs of chromosomes in humans. ... Chromosome 14 is one of the 23 pairs of chromosomes in humans. ... Chromosome 21 is one of the 23 pairs of chromosomes in humans. ... Chromosome 22 is one of the 23 pairs of chromosomes in humans. ...

Because modern microscopes can detect a range of colours in fluorescent dyes, by using whole-chromosome probe mixtures and a variety of colours, each human chromosome can be identified (M-FISH). There are currently twice as many chromosomes than fluorescent dye colours. However, ratios of probe mixtures can be used to create additional colours. Much like comparative genomic hybridization, the probe mixture for the secondary colours is created by mixing the correct ratio of two sets of differently-labeled probes for the same chromosome. Differently-coloured probes can be used for the detection of translocations. Several techniques exploit the resolution limitations of microscopes to resolve spatial distributions of dye below a few hundred nanometers. Colours that are adjacent appear to overlap, and a secondary colour is observed. Comparative genomic hybridization (CGH) is a molecular-cytogenetic method for the analysis of regional changes in the DNA content of tumor cells. ... A nanometre (American spelling: nanometer) is 1. ...

In reciprocal translocations, where both breakpoints are known, locus-specific probes are made for it and part of the region one either side of breakpoint. In normal cells, two colours will be visible; in diseased cells such as is found in BCR/ABL translocations, the two dye colours overlap, and a third colour is observed. This technique is known as double-fusion FISH or D-FISH. In translocations where only one of the breakpoints is known or constant, locus-specific probes are made for one side of the break point and the other intact chromosome. In normal cells, the secondary colour is observed but only the primary colour is observed when the translocation occurs. This technique is known as "break-apart FISH". Philadelphia chromosome or Philadelphia translocation is a specific genetic, chromosomal abnormality that is associated with chronic myelogenous leukemia (CML) and involves an exchange of material between chromosomes 9 and 22. ...

Medical applications

Often parents of children with a developmental delays want to know more about their child's conditions before choosing to have another child. These concerns can be addressed by analysis of the parents' and child's DNA. In cases where the child's developmental delay is not understood, the cause of it can be determined using FISH and cytogenetic techniques. Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia and Down syndrome. Mental retardation (abbreviated as MR), is a term for a pattern of persistently slow learning of basic motor and language skills (milestones) during childhood, and a significantly below-normal intellectual capacity as an adult. ... A metaphase cell positive for the bcr/abl rearrangement using FISH Cytogenetics is the study of the structure of chromosome material. ... Prader-Willi syndrome is a genetic disorder, in which seven genes (or some subset thereof) on chromosome 15 are missing or unexpressed (chromosome 15q partial deletion) on the paternal chromosome. ... Angelman Syndrome (AS) is a rare neuro-genetic disorder named after an English pediatrician, Dr. Harry Angelman, who first described the syndrome in 1965. ... Chronic myelogenous leukemia (CML) is a form of chronic leukemia characterized by increased and unregulated clonal production of predominantly myeloid cells in the bone marrow. ... Acute lymphoblastic leukemia (ALL), also known as acute lymphocytic leukemia, is a cancer of the white blood cells, characterised by the overproduction and continuous multiplication of malignant and immature white blood cells (referred to as lymphoblasts) in the bone marrow. ...

In medicine, FISH can be used to form a diagnosis, evaluate prognosis, or to evaluate remission of a disease, such as cancer. Treatment can then be specifically tailored. A traditional exam and metaphase chromosome analysis is often unable to identify features that distinguish one disease from another, due to subtle chromosomal features; FISH can elucidate these differences. FISH can also be used to detect diseased cells more easily than standard Cytogenetic methods, which require dividing cells and requires labor and time intensive manual preparation and analysis of the slides by a technologist. FISH, on the other hand, does not require living cells and can be quantified automatically, a computer counts the fluorescent dots present. However, a trained technologist is required to distinguish subtle differences in banding patterns on bent and twisted metaphase chromosomes. In general, a diagnosis (plural diagnoses) has two distinct dictionary definitions. ... Prognosis (older Greek πρόγνωσις, modern Greek πρόγνωση - literally fore-knowing, foreseeing) is a medical term denoting the doctors prediction of how a patients disease will progress, and whether there is chance of recovery. ... Remission is the state of absence of disease activity in patients with known chronic illness. ... Cancer is a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis (where cancer cells are transported through the bloodstream or lymphatic system). ... A metaphase cell positive for the bcr/abl rearrangement using FISH Cytogenetics is the study of the structure of chromosome material. ...

Species identification

FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria, from the patient's tissues or fluids, is typically grown on agar to determine the identity of the pathogen. Many bacteria, however, even well known species, don't grow well under laboratory conditions. FISH can be used to detect directly the presence of the suspect on small samples of patient's tissue. See drugs, medication, and pharmacology for substances that treat patients. ... A pathogen or infectious agent is a biological agent that causes disease or illness to its host. ...

FISH can also be used compare the genomes of two biological species, to deduce evolutionary relationships. A similar hybridization technique is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region. In biology, a species is a kind of organism. ... This article is about evolution in biology. ... A zoo blot is a DNA hybridization technique that demostrates the similarity between specific DNA sequences. ... A non-coding RNA (ncRNA) is any RNA molecule that functions without being translated into a protein. ...

FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms, for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, and can be useful in determining the fine architecture of the biofilm. Microbial ecology is the relationship of microorganisms with themselves and with their surroundings. ... A microorganism or microbe is an organism that is so small that it is microscopic (invisible to the naked eye). ... Longest raised mat area is about half a meter long. ...

See also

• In situ hybridization
• Molecular cytogenetics

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i. ... Molecular cytogenetics involves the combination of molecular biology and cytogenetics. ...

References

External links

• MeSH Fluorescent+in+Situ+Hybridization
• Information on fiber FISH from the Olympus Corporation
• A guide to fiber FISH from Octavian Henegariu
• Fibre FISH protocol from the Human Genome Project at the Sanger Centre