within the set of chromosomes. With the use of a
Fluorescence special microscope, the chromosome and
sub-chromosomal location where the fluorescent
bound probe can be seen. [1]
In-Situ
Hybridization
FISH is a molecular cytogenetic technique that
allows the localization of a specific DNA
sequence or an entire chromosome in a cell. It is
utilized to diagnose genetic diseases, gene
(FISH) mapping, and identification of chromosomal
abnormalities, and may also be used to study
comparisons among the chromosomes'
arrangements of genes of related species. [1]
Contents
● Introduction
● Timeline
● Principle
● Construction
● Probes
● Mechanism
● Protocol
● Calibration
● Data Analysis
● Types Fig 1: DNA bound to Fluorescent Probe
● Application
● Negative Aspects
● Conclusion
FISH involves unwinding of the double helix
● Use in a Research Paper
structure and binding of the DNA of all probes
● References
attached to a fluorescent molecule with a
specific sequence of sample DNA, which can be
Introduction visualized under the fluorescent microscope. [1]
Fluorescence in situ hybridization (abbreviated
FISH) is a laboratory technique used to detect Principle
and locate a specific DNA sequence on a
chromosome. In this technique, the full set of Fluorescence in situ hybridization (FISH) is a
chromosomes from an individual is affixed to a technique that uses fluorescent probes which
glass slide and then exposed to a “probe”—a bind to special sites of the chromosome with a
small piece of purified DNA tagged with a high degree of sequence complementarity to the
fluorescent dye. The fluorescently labeled probe probes. The fluorescent probes are nucleic acids
finds and then binds to its matching sequence labeled with fluorescent groups and can bind to
2
, specific DNA/RNA sequences. Thus, we can Probe Design and Labeling
understand where and when specific DNA
sequences exist in cells by detecting the ● Probe Design: Probes can be designed to
fluorescent group. Fluorescence microscopy can target specific genes, sequences, or
be used to find out where the fluorescent probe chromosomal regions. These include:
is bound to the chromosomes and flow ○ Single-stranded DNA (ssDNA)
cytometry can be used to detect the binding ○ RNA probes
quantitatively. [2] Key Steps Include : ○ Oligonucleotides
● Labeling: Probes are labeled with
● Probe preparation: Designing and fluorophores such as FITC, Cy3, or
labeling the probe with a fluorophore. Alexa Fluor dyes. Alternatively, haptens
● Sample preparation: Fixing cells or like biotin or digoxigenin can be used,
tissues on slides and denaturing the which require secondary detection.
DNA.
● Hybridization: Incubating the probe Sample Preparation
with the target DNA.
● Fixation: Tissue or cell samples are
● Washing: Removing unbound probes to
fixed to maintain cellular integrity and
ensure specificity.
preserve DNA.
● Detection: Observing the fluorescence
● Denaturation: Both the probe and target
signal under a microscope
DNA are denatured to form single
strands, usually by heat or chemical
treatment.
Probes
A variety of probes are used in the process of
FISH. Both double stranded and single stranded
probes are used which include cDNA, cRNA,
and other artificially synthesized oligonucleotide
probes. A probe is 50-300 base pairs in size. A
number of things are considered during making
Fig 2: Scheme of the principle of the FISH the choice of a probe such as - sensitivity,
experiment to localize a gene in the nucleus. specificity, the stability of hybrids, and ease of
application.
Generally, researchers use three different types
of FISH probes, each of which has a different
application:
Locus specific probes bind to a particular
region of a chromosome. This type of probe is
3
Fluorescence special microscope, the chromosome and
sub-chromosomal location where the fluorescent
bound probe can be seen. [1]
In-Situ
Hybridization
FISH is a molecular cytogenetic technique that
allows the localization of a specific DNA
sequence or an entire chromosome in a cell. It is
utilized to diagnose genetic diseases, gene
(FISH) mapping, and identification of chromosomal
abnormalities, and may also be used to study
comparisons among the chromosomes'
arrangements of genes of related species. [1]
Contents
● Introduction
● Timeline
● Principle
● Construction
● Probes
● Mechanism
● Protocol
● Calibration
● Data Analysis
● Types Fig 1: DNA bound to Fluorescent Probe
● Application
● Negative Aspects
● Conclusion
FISH involves unwinding of the double helix
● Use in a Research Paper
structure and binding of the DNA of all probes
● References
attached to a fluorescent molecule with a
specific sequence of sample DNA, which can be
Introduction visualized under the fluorescent microscope. [1]
Fluorescence in situ hybridization (abbreviated
FISH) is a laboratory technique used to detect Principle
and locate a specific DNA sequence on a
chromosome. In this technique, the full set of Fluorescence in situ hybridization (FISH) is a
chromosomes from an individual is affixed to a technique that uses fluorescent probes which
glass slide and then exposed to a “probe”—a bind to special sites of the chromosome with a
small piece of purified DNA tagged with a high degree of sequence complementarity to the
fluorescent dye. The fluorescently labeled probe probes. The fluorescent probes are nucleic acids
finds and then binds to its matching sequence labeled with fluorescent groups and can bind to
2
, specific DNA/RNA sequences. Thus, we can Probe Design and Labeling
understand where and when specific DNA
sequences exist in cells by detecting the ● Probe Design: Probes can be designed to
fluorescent group. Fluorescence microscopy can target specific genes, sequences, or
be used to find out where the fluorescent probe chromosomal regions. These include:
is bound to the chromosomes and flow ○ Single-stranded DNA (ssDNA)
cytometry can be used to detect the binding ○ RNA probes
quantitatively. [2] Key Steps Include : ○ Oligonucleotides
● Labeling: Probes are labeled with
● Probe preparation: Designing and fluorophores such as FITC, Cy3, or
labeling the probe with a fluorophore. Alexa Fluor dyes. Alternatively, haptens
● Sample preparation: Fixing cells or like biotin or digoxigenin can be used,
tissues on slides and denaturing the which require secondary detection.
DNA.
● Hybridization: Incubating the probe Sample Preparation
with the target DNA.
● Fixation: Tissue or cell samples are
● Washing: Removing unbound probes to
fixed to maintain cellular integrity and
ensure specificity.
preserve DNA.
● Detection: Observing the fluorescence
● Denaturation: Both the probe and target
signal under a microscope
DNA are denatured to form single
strands, usually by heat or chemical
treatment.
Probes
A variety of probes are used in the process of
FISH. Both double stranded and single stranded
probes are used which include cDNA, cRNA,
and other artificially synthesized oligonucleotide
probes. A probe is 50-300 base pairs in size. A
number of things are considered during making
Fig 2: Scheme of the principle of the FISH the choice of a probe such as - sensitivity,
experiment to localize a gene in the nucleus. specificity, the stability of hybrids, and ease of
application.
Generally, researchers use three different types
of FISH probes, each of which has a different
application:
Locus specific probes bind to a particular
region of a chromosome. This type of probe is
3
