Fluorescent in Situ Hybridization
Fluorescent in Situ Hybridization
Fluorescent in situ hybridization (FISH) is a powerful technique used to identify the presence of specific chromosomes or parts of chromosomes through the attachment (hybridization) of fluorescent DNA probes to available chromosomal DNA. The fluorescent DNA sequence used to attach to the cellular DNA is called the probe and is created in the experimental laboratory. Sometimes an RNA sequence is used as the probe instead of DNA. Examining the labeled cellular DNA under special lighting reveals the presence or absence of a fluorescent signal that indicates specific genes. FISH can be used on tissue preparations, blood or bone marrow smears, directly on cells, or on nuclear isolates.
In situ is Latin for "in the original place," which, in the case of FISH, means inside a human cell or tissue. To hybridize with something means to attach to it in a very selective, specific manner. In situ hybridization (ISH) is the attachment of a very specifically designed DNA probe to cellular DNA (the original place). FISH uses a DNA probe that can be labeled with a fluorescent compound, and emit colored light when it is exposed to specific light wavelengths under a microscope. FISH can detect specific DNA or RNA sequences that are present in a human cell, by taking advantage of DNA's double stranded nature.
FISH and DNA structure
In a normal human cell, DNA is compartmentalized in an area known as the cell's nucleus. Within this nucleus, the preferred conformation of DNA is two strands wrapped around each other and twisted. This twisted structure is known as the DNA helix. DNA is made up of chemical bases that are represented by the letters C, T, G, and A. This is the DNA alphabet that makes up each strand of DNA. The letters of each strand pair up in a specific manner when twisting to form the helix. All the T bases pair with A bases, and all the G bases pair with C bases. Different combinations of these bases are put together in three-letter "words." The arrangement of the words is what determines what a gene will encode for, give the gene its meaning, and therefore tell the body how to grow and develop. DNA is transcribed into RNA, the beginning of expression of DNA in a cell. To express the product that the gene is encoding, RNA is translated into proteins that function in many capacities for life.
FISH takes advantage of the tendency of DNA to form base pairs with its corresponding letters. The DNA inside a cell can be experimentally exposed and tempoarily unraveled from its helical structure. To denature DNA means to take the unraveling a step farther and undo or the bonds between the bases from the two strands. Once the single stranded bases are exposed, carefully designed DNA sequences that can be fluorescently labeled can be used to probe the cell's set of DNA or RNA. At specific temperatures and under standardized laboratory conditions, the probe is able to hybridize with (pair up with) and therefore label the cellular DNA. This technique can be used to search for specific gene sequences in human tissue that would cause clinical complications. FISH can also reveal the actual location of a DNA sequence on a chromosome .
The FISH technique
The general procedure for FISH involves fixing samples of chromosomes or human tissue onto a piece of glass known as a slide (it slides into place on the viewing platform of a microscope when the sample is ready to examine). To prepare the tissue on the slide for hybridization, it is treated with chemicals to permeabilize (open up) the cells and expose the DNA. The chemicals also denature the DNA so that it is single stranded and ready for the probe. A specific chemical hybridization solution containing the probe is applied to the slide so that the probe can hybridize with cellular DNA. This hybridization solution controls the degree of specificity to which the probe hybridizes to the target sequence. Factors such as the temperature, pH, and salt concentration can be changed to control the specificity of the hybridization. When the probe is made of RNA or is being hybridized to RNA, special precautions must be taken because single stranded RNA is less stable than DNA and easily degraded. Any excess probe is washed away.
Probes can be labeled directly with an attached fluorescent molecule, or indirectly, where a specific fluorescent-labeled antibody or labeled binding protein is used to detect a tag attached to the probe sequence. Using the indirect method, the probe itself only contains an attachment point for the fluorescent antibody or binding protein. With this method, the probe by itself is not fluorescent. Once the fluorescent binding molecules are applied, the slide can be viewed on a special microscope designed for fluorescence. The microscope applies a beam of light, set at a specific light wavelength to the DNA on the slide. The fluorescent tags on the DNA emit colored light in response to specific wavelengths. The fluorescent molecules do not fluoresce under sunlight.
Fluorescent compounds are known as fluorochromes. An example of a fluorochrome that may be used is fluorescein isothiocyanate (FITC). FITC can be attached, or conjugated, to an antibody for the tag on the DNA probe. FITC can only fluoresce under specific narrow wavelengths of light, and does not fluoresce in sunlight. Sunlight contains many wavelengths of light measured in nanometers (nm). Short wavelengths less than 400 nm are types of ultraviolet light that have very high energy. The visible spectrum of light ranges from 400–760 nm. The infrared, long wavelengths of light lie between 760–3,000 nm. FITC compounds are designed to only fluoresce when specific narrow ranges of wavelengths are shining on them. The wavelength required varies from compound to compound. A dark room and a special microscope equipped with such lighting are used for this purpose. Fluorescent labeling can allow two or more different probes to be visualized at the same time because they fluoresce with different colors and can be distinctly visualized. Special filters have been developed to allow simultaneous visualization of several fluorescent molecules at once. When many gene loci (locations) on a chromosome are being labeled, the process is referred to as a chromosome paint. Fluorescent dyes are subject to photobleaching (fading) and so are not permanent preparations. Digital imaging systems can store the fluorescent images permanently and make quantitative measurements.
Once the DNA has been visualized, many types of information can be revealed. FISH is an extremely powerful technique used for many applications in medical research and diagnosis. FISH can be used to determine chromosome structure, chromosome deletions, chromosomal gene mapping, detect the expression of genes when probing RNA, to localize viral DNA sequences, diagnose viral diseases based on the presence of viral DNA sequences, localize genes involved in cancer formation, in forensics, and in sex determination. There are so many uses and different approaches to FISH that it impacts many different types of medicine and research fields.
FISH applications in medicine
There are specific types of genetic disorders whose detection and diagnosis have been revolutionized by the FISH technique in accuracy, time, and cost. FISH findings have been determined as so important, that the standing committee for the International System on Cytogenetic Nomenclature (ISCN) established a specific genetic nomenclature just to describe FISH findings. Deletion syndromes, such as Prader-Willi syndrome , were first characterized by high-resolution analysis of chromosomes. Because the deletions in some of these disorders are small and difficult to detect, they are referred to as microdeletion syndromes. The FISH technique has revolutionized the detection and diagnosis of microdeletion syndromes. In some cases, the prevalence of these diseases had not been realized before FISH.
FISH has had a great impact on the characterization of chromosome structural abnormalities that are difficult to diagnose. Given a patient with a genetic disease that has multiple possible gene mutations , FISH, with multiple fluorochromes, can be used to determine the precise nature of the chromosomal rearrangements. FISH can be used to literally map out the exact chromosomal structure in DNA samples from such patients, to a level of accuracy previously unknown. This kind of diagnosis would not have been confirmed prior to the advent of FISH. FISH studies can also be used to screen for fetal aneuploidies (too many or too few of one type of chromosome), such as Down syndrome .
FISH is used in cancer analysis. Exploration of the acquired chromosomal abnormalities found in cancer cells is an important area of research. Techniques other than FISH usually require growing sample cells in laboratories for study, a task that can prove very difficult. Because FISH can be used on non-dividing cells, it can greatly augment standard research techniques. FISH studies are also being used to look for early relapse and residual disease in cancer patient bone marrow transplants from opposite sex donors. The success of transplant engraftments is monitored by dual fluorochrome FISH studies that can label and differentiate between the proportions of female XX and male XY cells in bone marrow and blood.
FISH is used in gene mapping of specific chromosomes and chromosomal regions. The DNA sequences within a chromosome can be determined by labeling FISH probes with multiple different fluorochromes and distinguishing their hybridization color patterns. Chromosomes are composed of DNA and associated proteins. The combination of DNA and protein found in chromosomes is called chromatin. In a new technique called fiber FISH, chromosome-specific chromatin fibers are stretched out on a glass slide and hybridized with gene locus-specific probes. Fiber FISH achieves higher levels of fine resolution mapping of DNA sequences than normal FISH.
FISH is a powerful technique. Because of its high accuracy, time efficiency, and relative low cost, it is quickly becoming the preferred method by which to accomplish many clinical and research applications.
Thompson & Thompson Genetics in Medicine, Sixth Edition. St. Louis, MO: Elsevier Science, 2004.
Heiskanen, M., O. Kallioniemi, and A. Palotie. "Fiber-FISH: Experiences and A Refined Protocol." Genet Anal. 12, nos. 5–6 (March 1996): 179–84.
Maria Basile, PhD