What Is Fluorescence In Situ Hybridization?
Fluorescence in situ hybridization (FISH), technique that employs fluorescent probes for the detection of unique deoxyribonucleic acid (DNA) sequences in chromosomes. FISH has a much better rate of sensitivity and specificity than different genetic diagnostic exams which includes karyotyping and consequently may be used to stumble on a variety of structural abnormalities in chromosomes, inclusive of small genetic deletions regarding just one to five genes. It is also beneficial in detecting slight-sized deletions consisting of those causing Prader-Willi syndrome, an extraordinary genetic disorder characterized through a rounded face, low forehead, and intellectual incapacity. FISH additionally gives outcomes more speedy than karyotyping due to the fact no mobile way of life is required.
FISH is commonly used for preimplantation genetic prognosis (PGD) in the course of in vitro fertilization. PGD entails acquiring an unmarried cell from an embryo inside the blastocyst degree of development. This unmarried mobile can then be analyzed using FISH. One problem with the usage of FISH for PGD is that an unmarried cell is scant cloth for diagnosis; consequently, a large array of assessments cannot be accomplished. Similarly, if the test fails for any technical cause, it can not be repeated.
The classical cytogenetics used trypsin-Giemsa or fluorescent banding pattern for identity and characterization of different chromosomal abnormalities inclusive of polycentric chromosomes, ring chromosomes, or chromatid interchanges. Though chromosome banding strategies based totally on Giemsa staining revolutionized cytogenetic analysis, they did no longer grow to be famous due to constrained decisions regarding handiest >three Mb of DNA. Certain chromosomal aberrations including reciprocal translocations and inversions had been not without difficulty recognizable with Giemsa stain. Besides that these techniques are very time consuming, and interpretation of karyotype could be very cumbersome and unsure.
In situ hybridization techniques initially advanced through Joseph Gall and Mary Lou Pardue in Nineteen Sixties (Pardue and Gall 1969) and John et al. (1969) have proved to be effective tools for figuring out the chromosomal location of hybridized nucleic acid. Soon after that fluorescent labels quickly replaced radioactive labels in hybridization probes because of their more protection, stability, and simplicity of detection.
Early in situ research used radioactive RNA or DNA probes that were categorized with 3H or 135I, and the sites of hybridization were detected by using autoradiography. These strategies were effectively implemented to both animals and plant life. RNA probes may be designed for any gene or any sequence inside a gene for visualization of mRNA, long noncoding RNA and miRNA in tissues and cells. These probes, often derived from the fragments of DNA that have been isolated, purified, and amplified for use in the Human Genome Project, consist of approximately 20 oligonucleotide pairs and cover an area of forty–50 bp of target RNA. In 1982, a new technique was described to localize DNA sequences hybridized in situ to chromosomes. This technique applied a biotin-labeled analogue of thymidine (TTP) which may be included enzymatically into DNA probes by means of nick translation. The web sites of hybridization have been detected either cytochemically by means of using avidin conjugated to horseradish peroxidase, or fluorometrically by using the usage of fluorescein-classified antibodies. Compared to autoradiography this method decreased the time required for detection, stepped forward decision, and gave less non-unique history and chemically solid hybridization probes.
Besides that non-isotopic strategies were developed the use of DNA probes labeled with amino acetyl fluorene (AAF), mercuration, and sulfonation, which can be detected after hybridization through affinity reagents. Recently a very effective machine has been described that uses digoxigenin-categorized nucleotides detected with the aid of antibodies sporting fluorescent or enzymatic tags. The non-isotopic labeling techniques have also been successfully applied for detection of extraordinarily repeated DNA sequences in plant chromosomes. The non-isotopic detection of low- or unmarried-replica genes, but, has no longer been a success.
Chromosome painting – aggressive hybridization using whole chromosomes – precise libraries for chromosomes as probes and human genomic DNA as the competitor became one of the first packages of FISH (Fig. 16.1). It supplied severe and particular fluorescent staining of the human chromosome in metaphase unfold and interphase nuclei. A translocation t(nine;22)(q34;p11) turned into first recognized in human neoplasia leading to Philadelphia chromosome.
Fluorescence in situ hybridization (FISH) is the most convincing method for locating the precise DNA sequences, prognosis of genetic illnesses, gene mapping, and identification of novel oncogenes or genetic aberrations contributing to various types of cancers. FISH involves annealing of DNA or RNA probes connected to a fluorescent reporter molecule with specific target collection of sample DNA, which can be observed underneath fluorescence microscopy. The approach has these days been increased to allow screening of the entire genome simultaneously thru multicolor entire chromosome probe strategies which includes multiplex FISH or spectral karyotyping or through an array-primarily based technique the usage of comparative genomic hybridization. FISH has absolutely revolutionized the sector of cytogenetics and has now been identified as a dependable diagnostic and discovery device within the fight in opposition to genetic diseases.
Here's how FISH works:
Preparation of Sample: First, a biological sample, such as cells on a microscope slide or tissue sections, is prepared for analysis. This may involve fixing the cells or tissue and permeabilizing them to allow the probe to enter the cells.
Probe Design: A FISH probe is designed. This probe is a small, single-stranded piece of DNA or RNA that is complementary to the target DNA sequence of interest. The probe is labeled with a fluorescent molecule, which gives it its name "fluorescence in situ hybridization."
Hybridization: The labeled probe is then applied to the sample and allowed to hybridize (bind) to the complementary DNA sequence within the cells. If the target DNA sequence is present in the sample, the probe will bind to it.
Washing: Unbound probe molecules are washed away to reduce background noise.
Detection: The sample is examined under a fluorescence microscope. If the target DNA sequence is present in the cells, it will emit fluorescence when exposed to the appropriate wavelength of light. This fluorescence is captured by the microscope and can be visualized as bright spots or signals within the cells or tissue.
FISH has several applications, including:
Gene Mapping: FISH can be used to map the location of specific genes on chromosomes, helping researchers understand gene organization and genetic mutations.
Cancer Diagnosis: It is used in cancer diagnostics to detect specific genetic abnormalities, such as gene amplifications or translocations, which can help determine the type and stage of cancer.
Prenatal Testing: FISH can be employed in prenatal testing to detect chromosomal abnormalities in developing fetuses.
Microbial Ecology: FISH is used in environmental microbiology to identify and quantify specific microorganisms in environmental samples.
Biomedical Research: It is a valuable tool for studying gene expression, chromosome structure, and genomic organization in various research fields.
Overall, FISH is a versatile and widely used technique in molecular biology and genetics for visualizing and analyzing specific DNA sequences in cells and tissues.
Types of Genetic Testing
Fluorescence In Situ Hybridization (FISH) is a powerful molecular biology technique that offers several benefits for various applications, particularly in the field of genetics and cytogenetics. Some of the key benefits of FISH include:
Visualization of Specific DNA Sequences: FISH allows researchers to target and visualize specific DNA sequences within a cell or tissue sample. This is invaluable for studying the location and distribution of genes, chromosomal abnormalities, and other genetic features.
High Sensitivity: FISH is highly sensitive and can detect even small amounts of target DNA sequences. This makes it suitable for identifying genetic alterations associated with diseases such as cancer and genetic disorders.
Quantitative Analysis: FISH can be used for quantitative analysis by measuring the intensity of fluorescent signals. This enables researchers to determine the copy number of a particular DNA sequence, which is essential for genetic diagnostics and research.
Chromosomal Aberration Detection: FISH is widely used in clinical cytogenetics to identify chromosomal abnormalities such as translocations, deletions, duplications, and aneuploidy. It is particularly valuable in diagnosing conditions like Down syndrome, leukemia, and certain congenital disorders.
Tissue Localization: FISH can help localize specific DNA sequences within tissues, allowing researchers to study gene expression patterns, identify the presence of pathogens like viruses, and examine the spatial organization of genetic material in the nucleus.
Clinical Diagnosis: FISH is a crucial tool in clinical diagnostics for identifying genetic abnormalities, determining cancer subtypes, and guiding treatment decisions. It can be used to tailor therapies for patients based on the genetic characteristics of their tumors.
Research Tool: FISH is widely used in research to study a variety of biological processes, including gene regulation, genomic stability, and the organization of genetic material within cells. It has contributed to numerous scientific discoveries.
Speed and Efficiency: FISH is a relatively fast and efficient technique compared to other methods like polymerase chain reaction (PCR) for certain applications. It can provide results within hours, making it useful in time-sensitive clinical settings.
Versatility: FISH can be adapted for different purposes by using different probes and experimental conditions. Researchers can design probes to target specific genes, RNA, or other nucleic acids of interest.
Multiplexing: FISH can be combined with other techniques, such as immunohistochemistry, to simultaneously visualize both genetic and protein markers within the same tissue sample. This allows for a more comprehensive analysis of biological specimens.
Overall, FISH is a versatile and essential technique with wide-ranging applications in genetics, genomics, and clinical diagnostics. Its ability to provide detailed information about DNA and RNA localization and quantification makes it a valuable tool for both research and medical purposes.