Full Form of FISH, Principle, types, Advantages 

Fish stands for Fluorescence in situ hybridization, used to diagnose the gene sequence on the chromosome. A small segment of DNA tagged by Fluorescence dye is known as a probe. This fluorescent probe is attached with a matching sequence on a specific chromosome. With the help of a microscope, the location of the attack probe can be seen on a chromosome or sub chromosome. This technique applied for both plants and  animals.

Since the first In Situ Hybridization was invested by Gall and Pardue in 1969.The studied that by the help of molecular hybridization determine the location of chromosome of hybridized nucleic acid. 

Mukai did this technique in plant chromosome  at 18S.26SrRNA on Oryza, Soybean, Aegilops, Hordeum chromosome.The FISH is very useful technique for stabilizing the specific DNA sequences, gene mapping identification of cancer. This technique modified the overall field of cytogenetics as a relevant diagnostic tool for genetic disease.

FISH Principle 

The FISH is based on the principle of hybridization of nuclear DNA of the interphase or metaphase of a cell, which is fixed on a microscopic slide. This probe is labeled with fluorophore directly or indirectly by heptane. This probe mixes with targeted DNA sequence after denaturation that initiates annealing for complementary DNA sequence. In a simple way, fluorescent probes bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. The fluorescence labeled probes are used for detection of the specific gene sequence. 

FISH Protocols-

The FISH protocol is completed in two sections. First one is Denaturation and Hybridization and the second one is Washing and Detection.

The equipments and reagents used in this technique that help to perform the technique-

FISH Components

There are three major components of FISH -

1. Sample 
2. Fluorescent Probe
3. Fluorescence microscope

FISH Sample Preparation

The sample preparation for FISH is completed in five steps that follow in the manner. The sample preparation is a very important step in this technique. 

•  Prepare the slide with chromosomes of metaphase or interphase nuclei.
• Use ethanol for dehydration.
• The temperature of 70 c denature the DNA.
• Labeled probe denature.
• Take the sample at 37 C for 5-15 hours for hybridization.

Fluorescent Probe

There are different types of probes used in FISH for detecting the DNA sequence, but every probe has different characteristics as well as different applications such as:

Locus Specific Probes, Alphoid or centromeric repeat probes, Whole chromosome probes.

• Locus Specific Probes-  LSP used for isolation of a small portion of gene and showing where the chromosome is located and how many copies of a gene within a particular genome. LSP binds on a particular site of the chromosome.
• Centromeric or Alphoid repeat probe- CRP present in the middle of each chromosome. This probe is used to find out the missing genetic material on a particular chromosome. CRP used in combination with locus specific probes.
• Whole Chromosome Probes - WCP binds with a different sequence rather than given chromosome length. These WCP are collections of smaller probes.

FISH Microscope

For visualization of DNA probe the phenomena of Fluorescent microscopy is light microscopy in rich lihe used as source at high pressure mercury vapor lamps, Tungsten lamps or Xenon lamps.  In Fluorescent microscope two types of filter use for seeing the samples such as Exciting Filter used to let a certain wavelength of light passes that pass from the sample which are already dyed by fluorescence dye and Barrier filter  used to allow the visible light passes so that it can visualize by eye or image can be captured.

The outcomes of FISH come in two different patterns. That is  depend on the type of method use for performing the technique-

1.  The result comes in a cell per unit pattern, if the evaluation is used manually.
2. The result comes on a relative volume or area basis by advanced microscopy techniques and digital image processing.

FISH types

The FISH is a very unique and general technique. There are different types of FISH that use variation in the sequence and probes are also labeled. The probes are also of two types that is : 

Cellular and Acellular. 

The probes are also important for Fluorescence in situ hybridization their size is depends on the type of specific chromosome because short length chromosome is hybridize only short length, so for long strands of DNA and RNA hybridized only a specific targeted sites or some nucleotides (often 15- 25, these probes are complementary from the targeted sequences. There are different types of hybridization that depends on the type of probe use for hybridization and also their location-

1. Single Molecule RNA FISH (smFISH)- This method is used for detecting and qualifying the mRNA and other RNA molecules that are long in size present in a thin layer of tissue sample. In smFISH the probes do not bind with sequence, this sequence does not reach at sufficient Fluorescence from the other one. 
2. Fiber FISH- This method of FISH is used for chromosomes attached at which they are straight lines. This is done by the applying mechanical shear along with the light of the sides, at the sides these cells are fixed and then lysed and then solution of purified DNA. The samples of Fiber FISH are only made in specialized labs, and use the technique routinely.
3. Q FISH - This technique is used only for length of telomere. Q FISH attach with peptide nucleic acid (PANs) which is artificially synthesized polymer line DNA or RNA. The PANs are used for measuring the intensity of fluorescence.
4. Flow FISH-  The flow FISH used to detect the physical and chemical characteristics of the population of cells and particles. This is an automatic technique that uses counting of fluorescent cells. 
5. MA FISH- The Microfluidics assisted Fluorescence in situ hybridization used to increase the efficiency of DNA hybridization by microfluidic flow. The MA FISH decreases the time of hybridization time and is less expensive. MA FISH used for detecting the Human Epidermal growth factor receptor (HER2)  
6. MARFISH- Multiplexed error Rebust Fluorescence in situ hybridization uses combinational labeling to capture a high number of RNA  molecules and septal localization in the cell.
7. STARFISH- The starfish is a type of software tool that is used for identifying the RNA molecule, removing distractions, and identifying the set of images and bioinformaticians. This tool developed in 2019 for determination of different variations of FISH.

FISH Application

The application of FISH is very vast and used in different area of Science such as:

Genetic counseling, Subcellular, Detect  and localize mutations on chromosome, species identification, biomedical research, Clinical diagnosis, Toxicology, Chromosomal biology, Comparative genomics.

FISH Advantages

The major role of FISH technology is that it determines the distribution of specific genetic material on protein products of the targeted gene and visualizes the hybridization at single cell level.

• Fast technique and increase the number of cells in a short time.
• Increase the sensitivity and specificity.
• Cytogenetic data can be obtained even from poor samples that contain few cells.
• Non dividing or terminally differentiated cells can also be obtained in cytogenetic data.

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