Used in genetics

1. Fluorescence in situ
hybridization (FISH)
Moderator – Dr Tharuni Latha
Presenter – Dr Shreyank H S

2. Contents
• Introduction
• Principle
• Metaphase and interphase FISH
• Steps involved in FISH
• Types of probes in FISH
• Advantages and disadvantages of FISH
• Diagnostic applications of FISH

3. Introduction
• In situ hybridization is the method of localizing/detecting specific
nucleotide sequences in morphologically preserved tissue sections or
cell preparations by hybridizing the complimentary strand of
nucleotide probe against the sequence of interest.
• DNA probes of a few hundred kilobases (kb) in length are used that
is complimentary to the regions of the chromosomes containing
the DNA sequence in question. These probes are directly hybridized
with the chromosomes on the slide (hence, the term "in
situ" hybridization).

4. • FISH combines conventional cytogenetics with molecular genetic
technology. It is based on the unique ability of a portion of single-
stranded DNA (i.e., a probe) to anneal with its complementary target
sequence on a metaphase chromosome, interphase nucleus or
extended chromatin fiber.

5. Principle involved in the FISH
• The basic principle involved is hybridization of nuclear DNA of either
interphase cells or of metaphase chromosomes affixed to a
microscopic slide, with a nucleic acid probe.
• The probes are either labeled indirectly with a hapten or directly
through incorporation of a fluorophore.
• The labeled probe and the target DNA are mixed together after
denaturation, which allows annealing of complementary DNA
sequences.

6. • In case the probe had been labeled indirectly, an extra step of
enzymatic or immunological detection system will be required for
visualization of the nonfluorescent hapten.
• The enzymatic detection system involves fluorochrome, which emits
colored signals at the hybridization site.
• The immunological detection system is based on binding of antibodies
to specific antigens, which is then demonstrated with a colored
histochemical reaction visible by light microscope or fluorochromes
with ultraviolet light

8. Metaphase and interphase FISH
• FISH can be performed on metaphase chromosomes or interphase
nuclei.
• Metaphase FISH allows identification of large
chromosomal abnormalities, including deletions, duplications, and
translocations, as well as smaller chromosomal microdeletions and
duplications.
• For metaphase FISH, cells are arrested in mitosis as for
chromosomal banding. They are then fixed using a mixture of acetic
acid and methanol and then “dropped” on a glass microscope slide
where they are affixed.

9. • Interphase FISH is used when dividing cells are not available (eg,
in fully differentiated cells or in tissues that have been fixed
and embedded in paraffin).
• For interphase FISH, cells are tested with FISH probes without
cell cycle synchronization.

10. • When using intact nuclei (eg, not from paraffin embedded tissue), the
cells are harvested using a hypotonic solution, fixed, and again
“dropped” on the slides. For FISH on paraffin embedded tissue, thin
slices of pathological specimens are cut and affixed to the slides.
• Because chromosomes are only minimally condensed in interphase,
this modification of FISH analysis provides the opportunity to
hybridize probes at a high resolution (well under 1 Mb, compared to 2
Mb for metaphase FISH).

11. • Advantages of interphase FISH include :
• The higher resolution than metaphase FISH.
• The ability to perform the test immediately without culturing the
cells, which makes it faster.
• The applicability to paraffin embedded sections.
• Interphase FISH is of special value in prenatal diagnosis of various
aneuploidy states, such as trisomy 18 or trisomy 21, in which the
ability to obtain results rapidly aids in decision making.

12. • Disadvantages of interphase FISH:
• The major disadvantage of interphase compared to metaphase FISH
is that in interphase FISH the chromosomes themselves cannot
be visualized. Thus, information cannot be provided regarding
overall chromosome number and composition.

13. Metaphase FISH Interphase FISH

14. Steps involved in FISH

15. Preparation of Probes :
• One of the most important steps in FISH analysis is the choice of
probe.
• A wide range of probes, extending from whole genomes to small
cloned probes (1–10 kb), can be used.
• There are basically three types of probes, each with a different range
of applications, whole chromosome painting probes, repetitive
sequence probes, and locus specific probes.

16. Types of FISH probes :
1] Centromeric Probes
• These consist of repetitive DNA sequences found in and around the
centromere of a specific chromosome. They were the original probes
used for rapid diagnosis of the common aneuploidy syndromes
(trisomies 13, 18, 21) using non-dividing cells in interphase obtained
from a prenatal diagnostic sample of chorionic villi.

17. 2] Chromosome-specific Unique-sequence Probes
• These are specific for a particular single locus. Unique sequence
probes are particularly useful for identifying tiny submicroscopic
deletions and duplications. Another application is the use of an
interphase FISH probe to identify HER2 overexpression in breast
tumors to identify patients likely to benefit from Herceptin treatment.

18. 3] Telomeric Probes
• A complete set of telomeric probes was been developed for all 24
chromosomes (i.e., autosomes 1 to 22 plus X and Y). Using these, a
method has been devised that enables the simultaneous analysis of
the subtelomeric region of every chromosome by means of only one
microscope slide per patient.

19. Probe Labeling :
• Several methods for labeling DNA probes for nonradioactive in situ
hybridization have become available.
• The most common approach is to label the probe with reporter
molecules (haptens). A variety of haptens are available in the market:
biotin, digoxigenin, dinitrophenol, fluorescein, rhodamine, AMCA, and
coumarin.
• These haptens can be incorporated as labeled nucleotides by tagging
technique of nick translation, random primer labeling, or PCR
according to the routine procedures.

20. • Detection of hybridized digoxigenin probes is mediated by
anti-digoxigenin antibodies conjugated to enzyme or fluorochrome.
• The labeled DNA may be separated from unincorporated
nucleotides using the spin column or ethanol precipitation methods.
The random primed labeling method is based on the hybridization of
a mixture of all possible hexanucleotides to the DNA to be labeled.

21. Examples

24. Advantages of FISH
• The resolution of FISH is much better than traditional chromosome
banding (FISH can resolve 2 megabases (Mb) in length, compared to 6
Mb for chromosomal banding).
• FISH can be applied to both dividing (metaphase) and non-dividing
(interphase) cells.
• The protocol is technically fairly straightforward.

25. • Hybridization with multiple probes enables detection of
translocation products. An example is the BCR-ABL1 fusion of t(9;22)
in chronic myeloid leukemia.
• FISH can identify a range of structural abnormalities
including deletions, duplications, aneuploidy and the presence of
derivative (structurally rearranged) chromosomes.
• FISH may be used to monitor recurrent or residual disease in
bone marrow transplant patients.

26. Disadvantages of FISH
• Small mutations, including small deletions and insertions as well as
point mutations, cannot be identified.
• Uniparental diasomy (inheritance of both copies of a chromosome
from the same parent) will be missed because the probe merely
detects the presence or absence of a locus or specific portion of a
chromosome and not its source.

27. • Chromosomal inversions will be missed since a probe can only detect
the presence of a specific sequence but not its precise location within
the chromosome.
• Probes are not yet commercially available for all chromosomal
regions.
• The clinician has to choose the correct FISH probe in order to make an
accurate diagnosis.

28. Diagnostic applications of FISH
• Prenatal diagnosis
• Cancer diagnosis
• Molecular cytogenetics of birth defects and mental retardation
• The identification of specific chromosome abnormalities
• The characterization of marker chromosomes
• Monitoring the success of bone marrow transplantation

29. References
• Emerys elements of medical genetics.
• Thompson and Thompson genetics in medicine.
• UpToDate
• PubMed

30. Thank you.