Embark on a journey into the microscopic world of genetics with our latest PDF, "Fluorescent In Situ Hybridization (FISH) Demystified"! 🧬✨ Join us as we unravel the intricacies of this groundbreaking molecular biology technique that allows us to visualize and understand genetic information at a whole new level. 🌈 Dive into the fascinating realm of genetics as we explore how FISH works, highlighting its crucial role in identifying and locating specific DNA sequences within cells. Witness the magic of fluorescent probes binding to target DNA, illuminating the intricate patterns that make up the blueprint of life. 🧫 Our expert molecular biologists guide you through the step-by-step process of conducting FISH experiments, from sample preparation to imaging. Gain insights into the applications of FISH across various fields, including cancer research, genetic diagnostics, and uncovering chromosomal abnormalities. 🎓 Whether you're a student, researcher, or simply curious about the wonders of molecular biology, "Fluorescent In Situ Hybridization (FISH) Demystified" provides a comprehensive and accessible overview of this revolutionary technique. 🎥 Don't forget to hit the subscribe button, give us a thumbs up, and ring the notification bell to stay updated on our scientific explorations. Delve into the world of genetic research with us – it's a journey that promises to illuminate the mysteries of life at the cellular level! 🔬🧪

1. Fluorescent In-Situ
Hybridization
(FISH)

2. Most important physical mapping
techniques
Restriction
mapping.
Fluorescent
in situ
hybridization
(FISH).
Sequence
tagged sites
(STS)
mapping.

3. Most important physical mapping
techniques
Restriction
mapping.
Fluorescent
in situ
hybridization
(FISH).
Sequence
tagged sites
(STS)
mapping.

4. What is the marker in this technique?
• In FISH the marker is a DNA
sequence that is visualized by
hybridization with a fluorescent
probe

5. Fluorescent In-Situ Hybridization
• Molecular cytogenetics technique allows the localization of a
specific DNA seq or an entire DNA
• Visualize the positions of a marker on a chromosome or extended
DNA molecule

6. The probe
radioactively labeled
• such as 32P or 35S
• detected using X-ray film or a
phosphorimager.
labeled with non-radioactive
fluorescent DNA labels
• Such fluorescent dyes
• high sensitivity with high resolution
and are ideal for in situ hybridization.
• Fluorolabels with different colored
emissions have been designed,
making it possible to hybridize several
different probes to a single
chromosome and distinguish their
individual hybridization signals,
enabling the relative positions of the
probe sequences to be mapped.
• detected using Fluorescence
Microscopy or Flow Cytometry

7. Cell Fixation: Cells are
first fixed onto a glass
microscope slide to
preserve their structure.
Permeabilization: The
cell membranes are
then permeabilized to
allow the entry of the
DNA probes.

8. Denaturation: The
chromosomal DNA is
denatured, meaning the
hydrogen bonds between
complementary DNA strands
are broken, and the double-
stranded DNA is converted
into single strands. This step
is crucial for allowing the
probes to bind to their
complementary sequences.
.

9. Hybridization: DNA
probes, labeled with a
fluorescent dye, are
then applied to the
denatured DNA on the
slide. These probes are
designed to be
complementary to
specific target
sequences in the
chromosomal DNA.

10. Washing: Excess and
unbound probes are
washed away.
Detection: The slide is
then examined under a
fluorescence microscope
to visualize the locations
where the probes have
hybridized to the
chromosomal DNA

11. prophase

12. prophase Metaphase

13. prophase Metaphase Anaphase

14. prophase Metaphase Anaphase Telophase

15. Advantages of metaphase FISH
• A fluorescent signal obtained by
FISH is mapped by measuring its
position relative to the end of the
short arm of the chromosome
• The main application of
metaphase FISH has been in
determining the chromosome on
which a new marker is located
and providing a rough idea of its
map position.

16. • The highly condensed nature of metaphase chromosomes means that
only low-resolution mapping is possible: 2 markers must be at least 1
Mb apart to be resolved as separate hybridization signals
• Metaphase FISH does not provide information about the organization
of chromosomes in non-dividing cells during interphase. Many
cellular processes, including gene expression and nuclear
architecture, are influenced by the spatial arrangement of
chromosomes in the interphase nucleus.
Disadvantages of metaphase FISH

17. Higher-resolution FISH techniques
have been developed
since 1995, by changing the nature of the
chromosomal preparation
and use more extended chromosomes

18. Non-metaphase chromosomes

19. Fiber-FISH
• Makes use of purified DNA prepared by
gel stretching or molecular combing can
distinguish markers that are less than10
kb apart
• Fiber-FISH provides higher resolution
compared to traditional metaphase FISH.
It allows for the direct visualization and
mapping of DNA sequences along
extended chromatin fibers, providing
information about the order and spacing
of genetic markers.