Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes to identify specific sequences on chromosomes. FISH allows researchers to detect locations of genes on chromosomes, identify chromosomal abnormalities, and determine copy numbers of genes. The FISH process involves preparing fluorescent probes with sequences complementary to the target sequence, hybridizing the probes to denatured chromosomes, and visualizing the fluorescent signals to identify the probe locations on chromosomes. FISH has various applications in gene mapping, diagnosing genetic disorders, and studying microbial communities in environmental samples.

1. FLUORESCENCE IN SITU
HYBRIDIZATION (FISH)
PREPARED BY – HAREKRUSHNA PRADHAN (40C/15)
2nd YEAR B.Sc. (Ag.)
SUBMITTED TO – DR. KAUSHIK KUMAR PANIGRAHI
ASST. PROFESSOR
PLANT BREEDING AND GENETICS

2. WHAT IS FISH ?
 Fluorescent in situ hybridization (FISH) is a molecular
cytogenetic technique that uses fluorescent probes
that bind to only those parts of the chromosome
with a high degree of sequence complementarity.
 It was developed by biomedical researchers in the
early 1980s.
 It is a technique used to detect the presence or
absence and location of specific gene sequences.
 FISH is a process which vividly paints chromosomes
or portions of chromosomes with fluorescent
molecules.

3.  It identifies chromosomal abnormalities & aids in
gene mapping, toxicological studies, analysis of
chromosome structural aberrations, and ploidy
determination.
 FISH looks specifically at the one specific area of a
chromosome only.
 A variety of specimen types can by analyzed using
FISH.
 The intact cells are attached to a microscope slide
using standard cytogenetic methods.
 A technique that hybridizes a DNA nucleic acid probe
to a target DNA sequence contained within a cell
nucleus.

4. Interpretation of FISH
 Each fluorescently labeled probe that hybridizes to a
cell nucleus in the tissue of interest will appear as a
distinct fluorescent dot
 Diploid nuclei will have two dots
 If there is duplication in the region of interest, the gain will
result in more than two dots
 If there is a loss in the region of interest, one or zero dot will
result
 If a small deletion is present in the region complementary to
the probe, the probe will not hybridise
 If a duplication is present, more of the probe is able to
hybridise.

5. Probes
Probe is a nucleic acid that
 can be labelled with a marker which allows identification and
quantitation
 will hybridize to another nucleic acid on the basis of base
complementarity
 A part of DNA (or RNA) that is complementary to certain sequence
on target DNA (i.e. DNA of the patient)
 Plasmid, phage DNA, cosmid (or combination of phage and
plasmid DNA
 PCR-product (amplification of certain segment of chromosomal
DNA)

6. TYPES OF PROBES
Scientists use three different types of FISH probes, each of
which has a different application
 LOCUS SPECIFIC PROBES
 ALPHOID or CENTROMERIC REPEAT PROBES
 WHOLE CHROMOSOME PROBES
 Locus specific probes bind to a particular region of a
chromosome. This type of probe is useful when scientists have
isolated a small portion of a gene and want to determine on
which chromosome the gene is located, or how many copies of a
gene exist within a particular genome.

7.  Alphoid or Centromeric repeat probes are generated from
repetitive sequences found in the middle of each chromosome. Researchers
use these probes to determine whether an individual has the correct number
of chromosomes. These probes can also be used in combination with "locus
specific probes" to determine whether an individual is missing genetic material
from a particular chromosome.
 Whole chromosome probes are actually collections of smaller
probes, each of which binds to a different sequence along the length of
a given chromosome. Using multiple probes labelled with a mixture of
different fluorescent dyes, scientists are able to label each chromosome
in its own unique colour. The resulting full-colour map of the
chromosome is known as a spectral karyotype. Whole
chromosome probes are particularly useful for examining chromosomal
abnormalities, for example, when a piece of one chromosome is attached to
the end of another chromosome.

8. How does FISH work?
 FISH is useful, for example, to help a researcher or clinician identify
where a particular gene falls within an individual's chromosomes.
 The first step is to prepare short sequences of single-stranded
DNA that match a portion of the gene the researcher is looking
for.
 These are called probes. The next step is to label these probes by
attaching one of a number of colors of fluorescent dye.
 DNA is composed of two strands of complementary molecules
that bind to each other like chemical magnets.

9.  Since the researchers' probes are single-stranded,
they are able to bind to the complementary
strand of DNA, wherever it may reside on a
person's chromosomes.
 When a probe binds to a chromosome, its
fluorescent tag provides a way for researchers to
see its location.

10. In which conditions we have to
indicate FISH analysis?
 The material doesn't contain metaphase chromosomes
 Analysis of complicated chromosomal rearrangements
 Identification of marker chromosomes
 Diagnosis of sub-microscopic (cryptic) chromosomal
rearrangements

11. Multi Colour FISH – A SPECIAL TYPE
Multicolour FISH can provide “colourized”
information relative to chromosome rearrangements,
especially useful in specimens where chromosome
preparations are less than optimal for standard
cytogenetic banding analysis

12. FISH Procedure
 Denature the chromosomes
 Denature the probe
 Hybridization
 Fluorescence staining
 Examine slides or store in the dark

15. Hybridization

16. Visualization of the Probe
 DNA probe is labelled with a coloured fluorescent
molecule.
 This fluorescent molecule remains attached to the
DNA during the hybridization process
 The molecule emits a particular colour when
viewed through a fluorescence microscope that is
equipped with the appropriate filter sets.

17. USES OF FISH
 Less labour-intensive method for confirming the presence of a
DNA segment within an entire genome than other conventional
methods like Southern blotting
 FISH method used in this study was suitable for the detection of
simazine-degrading bacteria and could be a useful indicator of
the potential of soil bioremediation.
 In environmental microbiology, FISH works have been carried out
with samples originated from sea water, rivers, lakes, biofilms,
soil, plants and animals.
 Fluorescent probes, like an intelligent stain, hybridize exclusively
with the rRNA of the chosen microorganisms allowing to:

18.  I) identify the microorganisms in environmental samples
without the utilization of culture media
 II) quantify the microorganisms directly in the sample
 III) determine the morphology
 IV) describe the spatial distribution
 V) determine the natural relation between species.

19. THANK
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