ISH and FISH

Hybridization
• Hybridization is a technique in which a double stranded DNA
molecule is formed in between a single stranded DNA probe
and a target single stranded DNA.
• The probes are labeled with a marker and complementary to
the target DNA.
• As a result we can detect one molecule of target in a
mixture of millions of molecules after hybridization, as the
reactions are specific.
 The Concept of In Situ Hybridization (ISH)

• “In situ” means “in the original place” in Latin. So ISH involves
a labeled nucleic acid probe hybridizing with a DNA sequence
in situ (in the cells).

• In situ Hybridization (ISH) is a method that allows to localize

and detect nucleic acid sequences within structurally intact
cells or morphologically preserved tissues sections.
 What is Fluorescence in
situ Hybridization (FISH)?
 What is fluorescence in situ hybridization
(FISH)?
• Early in situ studies used radioactive DNA probes that were
labeled with 3H or 135I, and the sites of hybridization were
detected by autoradiography.

• Soon after that fluorescent labels quickly replaced radioactive

labels in hybridization probes because of their greater safety,
stability, and ease of detection.

• Fluorescence in situ hybridization (FISH) is a kind of ISH

which uses fluorescent probes binding parts of the
chromosome to show a high degree of sequence
complementarity.
Principle of FISH
 Principle of FISH
The principles of fluorescence in situ hybridization:

(A)The basic elements are a DNA probe and a target sequence.

(B)Before hybridization, the DNA probe is labelled: either indirectly

with a hapten or directly labelled via the incorporation of a
fluorophore.

(C)The labelled probe and the target DNA are denatured to yield
single-stranded DNA.

(D)They are then combined, which allows the annealing of

complementary DNA sequences.

(E)Visualization of the target DNA with the help of labelled probe.

 Steps of FISH
1. Sample Collection and Fixation:

• Fresh tissue is collected and fixed.

• Fixation is done for preservation of tissues from decay due
to autolysis. It terminates any ongoing biochemical reactions and
may also increase the treated tissues' mechanical strength or
stability.

2. Embedding and Sectioning:

• Next, the sample is embedded in paraffin.

• Then the embedded tissues are sectioned into thin slices.
3. Permeabilization:

• The target DNA sequences are surrounded by proteins which

mask the target nucleic acid, and also present obstacles to good
infiltration of the probe.
• Reagents like proteases and detergents like Triton X-100 and
SDS are used to increase the permeability.

4. Denaturation:

• This is done to make the dsDNA into single strands so that the
probe can bind to its complementary sequence.
5. Hybridization:

• The probe now anneals with a complementary sequence of

the DNA strand.

6. Washes:

• Unbound or loosely bound probes are removed by performing

washes.

7. Detection:
There are two methods of probe detection:
1. Direct
2. Indirect
1. Direct methods:

• The detectable reporter i.e. the

fluorophore is bound to the
probe directly so that the
probe-target hybrids can be
detected under a microscope
immediately post-hybridization
washes.

• FISH is faster with direct

methods.
2. Indirect methods:

• If the probe has been labeled

indirectly, an extra step is
required for visualization of the
non-fluorescent probe that uses
an enzymatic or immunological
detection system.

• Indirect labeling offers the

advantage of signal amplification
by using several layers of
antibodies, and it might therefore
produce a signal that is brighter
compared with background
levels.
 Applications of FISH
• In FISH, the labelled DNA probe are allowed to bind to a person’s
DNA, corresponding to a particular genomic region of interest.
This is useful to assess chromosome or gene copy number.

• If there has been a duplication, more of the probe will hybridize

with the sample DNA and more fluorescence will be detected.

• If there has been a deletion in the genome, the probe cannot

attach to its complementary region as it is absent and no
fluorescence will be detected.

• Resolution of very small deletions (microdeletions) and small

translocations is possible with FISH.
Prenatal diagnosis of chromosomal abnormalities
• Prenatal diagnosis is critical to determine the health and
congenital abnormalities in the unborn fetus.
• Common chromosomal abnormalities include Edward’s syndrome
(presence of an extra copy of chromosome 18), and Down’s
syndrome (presence of an extra copy of chromosome 21).

Down’s syndrome
Edward’s syndrome
 Prenatal diagnosis of chromosomal
abnormalities
• When a florescent DNA probe is tested against healthy regions of
chromosome 18 or 21, two fluorescent signals corresponding to
two chromosomes would be observed. However, in the case of
presence of extra copy of chromosome, three florescent signals
would be visible.

Fetus with Trisomy-21

Normal fetus
Fig: Nuclei from amniotic fluid cells have been combined with DNA probes for chromosomes
13 and 21.The nucleus has been hybridized to probes for chromosomes 13 (green), and 21
(red).
 Therapy selection for breast cancer

Sometimes changes in a gene number can make the cancer

cells:

•make far more of the protein than normal

•stop making a particular protein

This can make the cancer cells grow and reproduce more than
normal.

Let’s take the example of breast cancer:

 Therapy selection for breast cancer

1. Human Epidermal Growth Factor Receptor 2 (HER2) is a

protein which helps breast cells grow and divide, whether
cancerous or normal.
2. The HER2 gene makes HER2 proteins.
 Therapy selection for breast cancer
• In about 10% to 20% of breast cancers, there are extra copies
of HER2 gene (known as HER2 gene amplification).
• All these extra HER2 genes lead to overexpression of the
HER2 proteins, up to 100 times as many. This makes breast
cells grow and divide in an uncontrolled way.
• Breast cancers with HER2 gene amplification or HER2 protein
overexpression are called HER2-positive in the pathology
report.
• HER2-positive breast cancers tend to grow faster and are more
likely to spread and come back compared to HER2-negative
breast cancers.
• Hence medicines specifically for HER2-positive breast cancers
should be given to these HER2-positive patients.
 Therapy selection for breast cancer

Figure: Representative view of breast carcinoma cells. HER2 signal

is represented in red; centromere 17 probe (green) can be used to
enumerate chromosome number.

A. HER2-non-amplified breast carcinoma: two centromeres 17 and

two copy of the HER2 gene as expected.
B. HER2-amplified breast carcinoma multiple detection for HER2.
 Therapy selection for breast cancer

• It is important to have an accurate HER2 status result in order

to treat HER2-positive breast cancer as effectively as possible.

• This includes the option of targeted therapies such as

trastuzumab, pertuzumab, lapatinib, and neratinib—drugs that
specifically address this protein.

• The particular types of chemotherapy for breast cancer that

work best can also vary with HER2 status.

• For patients who do not over-express HER2, these medications

are useless and they simply do not work.
 FISH over Karyotyping?
FISH technology offers three major advantages:

• High sensitivity and specificity in recognizing targeted DNA

• Direct application to both metaphase chromosomes and interphase
nuclei
• Visualization of hybridization signals at the single-cell level
• No need of cell culture and cell synchronization
• Higher resolution

However, it should be noted that false positive or negative results as

well as maternal cell contamination have been noted in prenatal FISH
analysis.
Therefore, an irreversible therapeutic action should not be initiated on
the basis of FISH results alone.
The current guideline recommended that clinical decisions should be
made based on two of three pieces of available information: FISH
results, conventional cytogenetic analysis and clinical information.