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Fluorescence in situ hybridization (FISH)

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Also listed as: FISH
Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • Amplification, cancer, chromosome, chromosome painting, colcemid, cytogenetic technique, deletion, disease, DNA, FISH, fluorescence, karyotype, microscope, mRNA, probe, RNA.

Background
  • Fluorescence in situ hybridization (FISH) is a method that researchers may use to determine whether a specific part of a chromosome is present in a cell. Chromosomes are located in a compartment of the cell called the nucleus and are composed of DNA and proteins. Human cells contain 46 chromosomes (23 pairs), and each chromosome has hundreds of genes. Genes contain the instructions for making the proteins that do the work in the human body.
  • To perform FISH, researchers may first identify a specific region of a chromosome that they are interested in studying (for example, a region that contains a specific gene). They then generate a probe, or a sequence of DNA, that can recognize and bind to the chromosomal region of interest. In FISH, the probes that researchers use are fluorescently labeled so that they emit a colored light if the chromosomal region of interest is present in a cell.
  • In some diseases (such as cancer), genetic mutations occur that can cause part of a chromosome to be deleted, repeated, or reversed in orientation. By looking for differences in how chromosomes from different cells stain with a probe, researchers may be able to observe these changes under the microscope and learn more about the genetic mutations that cause a particular disease.

Methods
  • To perform fluorescence in situ hybridization (FISH), researchers typically need to carry out several steps.
  • Obtain cells: First, a line of cells that a researcher is interested in studying are grown in the laboratory. Cell lines made from blood, amniotic fluid, or bone marrow are commonly used for analysis. After the cells have grown in number, a chemical (such as colcemid) is used to stop the cells from growing further. Colcemid inhibits the growth of cells when they reach a stage of the cell cycle called metaphase, when the chromosomes are easily visible. Colcemid stops the cells from growing by interfering with the function of microtubules (proteins needed by the cell for growth).
  • Prepare cells for observation: Once the cells have been obtained, the cells are exposed to a hypotonic solution (a solution that has a relatively low concentration of dissolved molecules), which expands the cells and spreads the chromosomes out, so they will be easier to examine. The cells are killed (using a fixative, such as methanol and acetic acid), and then transferred to a microscope where the chromosomes can be observed. Some of the metaphase cells are dropped onto a microscope slide (a thin piece of glass) and then spread apart on the slide with a small tip. They are then dried and they become attached to a slide.
  • Obtain probe: To perform FISH, researchers may first identify a region of a chromosome that they are interested in studying (for example, a region that contains a specific gene). They then generate a probe or a sequence of DNA that can recognize and bind to the chromosomal region of interest. Researchers can obtain the DNA material for a probe in several different ways. For example, they may perform a technique called chromosome microdissection, in which they physically remove part of a chromosome and then use it as a probe. Or they may obtain a piece of DNA to use as a probe from a DNA library (a DNA library is a large collection of isolated and purified DNA fragments that is frequently used in DNA sequencing projects). In some cases, a researcher may be interested in just detecting one specific chromosomal region, so only one probe will be used. However, in some cases, a researcher may want to study all the chromosomes at once. In these cases, researchers can make probes specific to each chromosome and use them all at once to broadly search for chromosomal abnormalities.
  • Apply probe to cells: Using the probe, researchers can determine whether the cells they are studying contain the region of DNA that the probe was designed to detect. The cells (which have been attached to microscope slides) are heated, so that the DNA will be more accessible to the probe. The probe is then added to the slide. If the chromosomal region of interest is present, the probe will bind to the chromosome in that region. After the probe has been given time to bind to the DNA (this may take several days), the cells are washed, so that the probe only remains in cells where it has detected and bound to a chromosomal region.
  • In FISH, the probes are fluorescently labeled so that when viewed with a fluorescent microscope, they emit a colored light if the chromosomal region of interest is present. When observed under the microscope, FISH probes often appear as small colored dots when they are bound to a chromosome. In some cases, researchers may choose to use more than one probe at the same time in a FISH experiment. Different colored FISH probes are available so that in an experiment, one probe could appear as a red dot and another probe could appear as a blue dot.

Research
  • Using fluorescence in situ hybridization (FISH), researchers can check for differences in the chromosomes between two different cells. In some cases, FISH may be able to demonstrate that one cell has an extra copy of a specific chromosome. In other cases, FISH may be able to detect smaller variations in a particular chromosome, such as a repeated region in the chromosome or a deletion in the chromosome. FISH is often used to study different types of diseases, such as cancer, in which mutations occur that cause extra copies of chromosomes to be present or that cause a portion of a chromosome to become deleted, repeated, or reversed in orientation.
  • FISH may be used to study evolutionary relationships between different species. If a probe is designed to detect a specific chromosomal region in one species, that probe can also be used to check for the same chromosomal region in another species. If the species are closely related, the probe will also bind to the chromosome of the other species; however, if the species are distantly related, the probe will no longer be able to bind to the chromosome of the second species. FISH may also be used to look for other changes that occur between two species during evolution, such as amplification of a particular chromosomal region.
  • In some cases, FISH may not be used to detect a region on a chromosome, but rather used to check for the presence of RNA. RNA is a molecule made from DNA, and RNA is used by a cell to produce proteins. RNA FISH is performed by generating a probe to detect a specific RNA, and then allowing that probe to hybridize, or bind, to the RNAs in a cell. This technique allows researchers to determine whether a specific RNA is produced by a cell and where in the cell that RNA is located.

Implications
  • Diagnosis: Fluorescence in situ hybridization (FISH) can be used to diagnose certain human diseases prenatally, before a baby is born. For example, diagnosis of genetic diseases such as trisomy 18 or trisomy 21 (also known as Down syndrome) may be performed on an unborn baby through amniocentesis, in which the amniotic fluid surrounding the unborn baby is sampled through a needle. Cells in the amniotic fluid can be used to perform FISH and check for an extra copy of chromosome 18 (in the case of trisomy 18) or an extra copy of chromosome 21 (in the case of Down syndrome).
  • FISH may also be used by doctors to diagnose diseases in children or adults. For example, Prader-Willi syndrome is a genetic disease in which part of chromosome 15 is deleted. Using FISH, doctors can design probes for chromosome 15 and detect the chromosome 15 deletion in patients. FISH may also be used to detect genetic diseases in which part of a particular chromosome has undergone an expansion. For example, in the neuromuscular disease myotonic dystrophy, part of chromosome 3 is duplicated many times.
  • Bacterial detection: Although in many cases FISH is used to analyze a cultured line of cells, FISH can also be used directly on bacteria cells that are not dividing to detect a bacterial infection. By generating probes that are specific for different strains of bacteria, doctors can detect whether that strain of bacteria is present in a patient's blood using FISH. For example, FISH has been used to detect the bacterial pathogen Pseudomonas (a cause of urinary tract infections) in blood samples.
  • Cancer prognosis: FISH may be used by doctors to determine a prognosis for some types of cancer. Using FISH, researchers have found that in prostate cancer cells, a specific gene (called the epidermal growth factor receptor) is present in many more copies than in normal cells. Genetic changes have been observed using FISH in other types of cancer as well, such as cervical cancer and breast cancer. These genetic changes may be used by doctors to diagnose cancers or to make a prognosis for the disease.
  • Understanding disease: FISH can be used to better understand some diseases. By understanding the specific rearrangements that chromosomes have undergone in patients, researchers can better understand the disease. This is because rearrangements may cause specific genes to become deleted or amplified, and the genes that have increased or decreased in number may play a role in causing the disease. By identifying these genes, scientists can better understand the cause of the disease, and they may be able to use this information to develop drugs to fight it. This approach is useful for the study of some types of cancer, in which chromosomes become mutated and undergo rearrangements.

Limitations
  • One drawback of fluorescence in situ hybridization (FISH) is that in order to generate a probe to detect a specific region of a chromosome, some information about the DNA sequence of that chromosome must already be known. It is not always necessary to know the specific DNA sequence of the area that is being examined. Researchers sometimes generate probes based on areas near the chromosomal regions of interest.
  • Researchers must also be careful when generating a probe. Different chromosomes may have similar chromosomal regions. Therefore, researchers may need to generate a probe that is specific to just the region they are interested in, but that is not similar to other pieces of the chromosome.

Safety




Future research
  • Not Applicable

Author information
  • This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography
  1. Bayani J, Squire JA. Fluorescence in situ hybridization (FISH). Curr Protoc Cell Biol. 2004 Sep;Chapter 22:Unit 22.4.
  2. Cho KS, Lee JS, Cho NH, et al. Gene amplification and mutation analysis of epidermal growth factor receptor in hormone refractory prostate cancer. Prostate. 2008 Feb 26.
  3. Mark HF, Feldman D, Das S, et al. HER-2/neu oncogene amplification in cervical cancer studied by fluorescent in situ hybridization. Genet Test. 1999;3(2):237-42.
  4. Moatter T, Khilji Z, Murad F, et al. Analysis of amniotic fluid specimens for common chromosome disorders using interphase fluorescence in situ hybridization. J Pak Med Assoc. 2007 Apr;57(4):189-92.
  5. National Human Genome Research Institute (NHGRI). .
  6. Natural Standard: The Authority on Integrative Medicine. .
  7. Suzuki Y, Sasagawa I, Yazawa H, et al. Detection of chromosome 15 deletion in Prader-Willi syndrome using fluorescence in situ hybridization. Arch Androl. 2000 Jul-Aug;45(1):13-7.
  8. The University of Hong Kong. .
  9. Yale School of Medicine. .

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