Fluorescence in-Situ Hybridization (FISH)

1. FLUORESCENCE IN
SITU HYBRIDIZATION
(FISH)
PREPARED BY – SHARADA PRASANNA SAHOO (23C/15)
2nd YEAR B.Sc. (Ag.)
SUBMITTED TO – DR. KAUSHIK KUMAR PANIGRAHI
ASST. PROFESSOR
( PLANT BREEDING AND GENETICS)

2. WHAT IS FISH ?
 Fluorescennce 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

7. 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.

8.  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.

9. 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

10. 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

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

14. Hybridization

15. 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.

16. 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:

17.  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.

18. THANK YOU