Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed in the early 1980s. FISH uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in colored signals that can be detected using a fluorescent microscope

1. FLUORESCENCE IN
SITU HYBRIDIZATION
Presented by:
Group # 05
Ayesha Ashraf (226208)
Saba Ashraf (226213)

2. Outlines
Introduction
Principle
Protocol
Trouble shooting
Results

3. Introduction
Fluorescence in situ hybridization (FISH) is used for the visualization
of specific DNA sequences in metaphase chromosomes and interphase
cells.
Chromosomal translocations, deletions, amplification of specific
genes and changes in chromosome number can all be detected by
using probes.
It provides increased sensitivity, in that cytogenetic abnormalities
have been found in samples that appeared to be normal by
morphologic and conventional cytogenetic examination.

4. Principle
When
two complementary
sequences find each
other thy bind together
are hybridized.
Fish works by
exploiting the ability of
one DNA strand to
hybridize specifically to
another DNA strands.

6. Requirements
1. Sample could be
I.DNA
II.RNA
2. Probes
3. Fluorescence Microscope

7. Types of Probes
1.
Centromeric
probe
2. Whole
Chromososme
3. Telomeric
probe
4. Gene
Specific probe
- Alpha & satellite
probes
- Generated from
repetitive sequence
found in centromere
- Centromere regions
are stained brighter
- Set of probes that
bind to whole
chromosomes
- Multiple probes
labels are used
to hybridize the length
of chromosomes
- Specific for
telomeres
- Specific to 300kb
locus at the end of
specific chromosomes
•Locus Deletion
•Translocation probes
•Gene detection and
localization probes
•Gene amplification
probes

8. Characteristics of
probes
1. Ds DNA probes- Stable, easily available
2. ss DNA probes - Stable, easier handling,
specific
3. RNA probes- Higher thermal stability, better
tissue penetration, more specific
4. Synthetic oligonucleotide probes- Economical,
stable, easier to handle, resistance to RNAse ,
better tissue penetration & better reproducibility

9. Methods of Labelling
1. Direct Labelling-
fluorescent nucleotides
are used
2. Indirect Labelling- is
incorporated with reporter
molecules that are
subsequently detected by
fluorescent antibodies or
other affinity molecules

10. Working of FISH
Denaturation
Sample
Preparation
Hybridization
Wash &
counter stain
Microscopic
Examination
1. 2. 3. 4. 5.

11. FLUORESCENCE MICROSCOPE
Optical instrument that use fluorescence to
generate an image
Principle
The specimen is illuminated with light of specific
wavelength which is absorbed by the fluorophores
causing them to emit light of longer wavelength.

12. Advantages
1.Rapid technique, and large numbers of cells can be scored in a short period
2. Efficient, Sensitivity and specificity is high.
3. Cytogenetic data can be obtained from nondividing cells.
4. Cytogenetic data can be obtained from poor samples that contain few cells.
5.Technique permits direct correlation of cytogenetic and morphologic features, enabling
pathologists to differentiate malignant from benign conditions in equivocal cases.
6. Method has been adapted for automated systems (useful for some probes, such as
centromere-specific probes).

13. Limitations
1.Restricted to those
abnormalities that can be
detected with currently
available probes.
2. Only one or a few
abnormalities can be
assessed simultaneously.
3. Cytogenetic data can be
obtained only for the
target chromosomes.
4. Requires fluorescence
microscopy and an image
analysis system.

14. Recent
Advancement
promising advances include
peptide nucleic acid probes (DNA
analogs in which the deoxyribose
has been replaced by 2-
aminoethyl glycine), which have
very rapid hybridization kinetics.
SKY and Rx-FISH now provides
the power to analyze the entire
genome in a single hybridization
experiment

15. Applications
1. Morphology: morphology and population structure of
microorganism
2. Pathology: pathogen profiling, abnormal gene expression
3. Developmental biology: gene expression profiling in
embryonic tissue
4. Karyotyping and phylogenetic analyusis: unique fish pattren
on individual chromosomes, chromosomal aberrations
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16. Thank You!!!