Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used to detect specific DNA sequences in chromosomes, primarily through fluorescent probes. Established in the 1980s, it has evolved to aid in clinical diagnostics, cancer research, and genetic studies by visualizing chromosomal abnormalities and gene expression. While offering high spatial resolution and multiplexing capabilities, FISH also faces challenges such as detecting small genetic alterations and requiring specialized resources.

1. MR. R. SUSIL
ASSISTANT PROFESSOR
GANGA INSTITUTE OF HEALTH SCIENCES
MEDICAL LABORATOTECHNOLOGY

2. Fluroscent Insitu Hybridation Techniques

3. Introduction to FISH Techniques
FISH is a molecular cytogenetic technique used
to detect and localize specific DNA sequences
in chromosomes.
This method employs fluorescent probes that
bind to specific sequences, allowing
visualization under a fluorescence microscope.
FISH is commonly used in genetic research,
clinical diagnostics, and cancer studies.

4. Historical Background
FISH was first developed in the 1980s as a
method to visualize chromosomal
abnormalities.
The technique has evolved significantly with
advances in fluorescence microscopy and
probe design.
It has become a standard tool in cytogenetics
and molecular biology.

5. Basic Principles of FISH
FISH relies on the hybridization of
fluorescently labeled probes to
complementary DNA sequences in fixed
cells.
The binding of the probes is followed by
washing steps to remove non-specific
binding.
Visualization is achieved through
fluorescence microscopy, where specific
colors indicate the presence of targeted
sequences.

6. Probes Used in FISH
Probes can be made from various
sources, including plasmids, PCR
products, or synthetic oligonucleotides.
They are labeled with different
fluorophores to allow the simultaneous
detection of multiple targets.
The choice of probe is crucial for the
specificity and sensitivity of the assay.

7. Sample Preparation
Sample preparation is critical for
obtaining high-quality results in FISH.
Cells or tissue sections must be fixed and
permeabilized to allow probe access to
DNA.
Proper fixation preserves the cellular
morphology and genetic material.

8. Hybridization Process
The hybridization step involves
incubating the prepared sample with
labeled probes under controlled
temperature and conditions.
Optimal hybridization conditions
depend on the probe and target
sequence characteristics.
After hybridization, the sample is
washed to remove unbound probes.

9. Detection and Visualization
Detection of hybridized probes is achieved using a fluorescence
microscope equipped with specific filters.
Different fluorophores emit light at distinct wavelengths, allowing for
multiplexing.
The intensity and location of fluorescence provide information about the
target sequences.

10. Applications in Clinical Diagnostics
FISH is widely used in clinical settings
for the diagnosis of genetic disorders
and cancers.
It helps identify chromosomal
abnormalities such as deletions,
duplications, and translocations.
FISH can guide treatment decisions
and prognostic evaluations in
oncology.

11. Applications in Research
In research, FISH is utilized to study gene
expression patterns and chromosomal
organization.
It aids in mapping the locations of
specific genes on chromosomes.
Researchers also use FISH for
evolutionary studies to examine
chromosomal changes across species.

12. Advantages of FISH
FISH provides high spatial resolution and can visualize specific sequences
in interphase and metaphase cells.
It allows for the analysis of multiple targets simultaneously, enhancing
throughput.
The technique is relatively quick compared to traditional karyotyping
methods.

13. Limitations of FISH
FISH may have limitations in detecting
very small genetic alterations due to
probe size constraints.
The technique requires specialized
equipment and trained personnel for
accurate interpretation.
FISH can be expensive, particularly
when using multiple probes or
advanced imaging systems.

14. Types of FISH Techniques
There are several types of FISH, including conventional FISH,
comparative genomic hybridization (CGH), and multicolor FISH (M-
FISH).
Each type serves specific purposes, such as detecting chromosomal
imbalances or visualizing multiple chromosomes simultaneously.
The choice of FISH technique depends on the research or clinical
question at hand.

15. Multi-Color FISH Techniques
Multi-color FISH employs multiple probes simultaneously to visualize several
targets in a single sample.
It allows for the detection of complex chromosomal rearrangements and
gene fusions.
The analysis of multi-color FISH requires advanced imaging techniques and
software.

16. Quantitative FISH (Q-FISH)
Q-FISH is a quantitative modification of
traditional FISH that provides
numerical data on target sequences.
It is useful for measuring gene copy
numbers and assessing structural
variations.
This technique enhances the
understanding of gene dosage effects
in various conditions.

17. FISH in Cancer Research
FISH plays a critical role in cancer
research by identifying genetic
alterations associated with
tumorigenesis.
It helps in the classification and
prognosis of different cancer types
based on chromosomal abnormalities.
FISH is also used to monitor treatment
response and detect minimal residual
disease.

18. FISH in Prenatal Diagnosis
FISH is employed in prenatal
diagnostics to detect chromosomal
abnormalities in amniotic fluid or
chorionic villus samples.
It allows for rapid results, which is
crucial for timely clinical decision-
making.
FISH can identify conditions like Down
syndrome and other aneuploidies.

19. Future Directions in FISH Technology
Advances in probe design, imaging techniques, and computational
analysis are enhancing FISH capabilities.
New developments aim to improve sensitivity and specificity for detecting
small genetic changes.
Future applications may include integration with next-generation
sequencing technologies.

20. Troubleshooting in FISH
Common issues in FISH include
nonspecific binding, low signal
intensity, and background
fluorescence.
Optimizing hybridization and washing
conditions can help mitigate these
problems.
Proper controls and replication are
essential for reliable interpretation of
results.

21. Conclusion
FISH is a versatile and powerful technique for the analysis of genetic
material in various fields.
Its applications continue to expand, driven by technological
advancements.
Understanding the principles and techniques of FISH is crucial for
researchers and clinicians alike.

22. THANK YOU