Fluorescent In Situ Hybridization

(FISH)

MUHAMMAD TARIQ
Medical Technologist, Dept of Blood Bank (SBT)
HMC Peshawar
M Sc. (Medical Technology). PhD. Fellow (Genetics)
 Fluorescent In Situ Hybridization
(FISH)

Fluorescence In Situ Hybridization

(FISH)
 FISH
• FISH (Fluorescent In Situ Hybridization) is a cytogenetic
technique which can be used to detect and localize the
presence or absence of specific DNA sequences on
chromosomes.
• It uses fluorescent probes which bind only to those parts
of the chromosome with which they show a high degree
of sequence similarity.
 FISH
• Fluorescencet microscopy can be used to find out where the
fluorescent probe bound to the chromosome.
• FISH is often used for finding specific features in DNA. These
features can be used in genetic counseling, medicine, and
species identification.
 ADVANTAGES OF FISH OVER
CHROMOSOME ANALYSIS
• A variety of specimen types can by analyzed
using FISH.
• The most powerful application is on formalin
fixed dead cells.
• Time duration of procedure is less than
conventional chromosome analysis.
 (FISH) TO RULE OUT:
 Chromosome Microdeletion Detection
 Interphase Chromosome Enumeration
 Gene Rearrangements (ie, bcr/abl, PML/RARA)
 Cryptic Chromosomal Rearrangements
 Marker Chromosome Identification
 Chromosome Breakpoint Mapping
 PRINCIPLE
• FISH technique utilize the phenomenon of
fluorescence to identify specific sequence on a
chromosome or a chromosome as a whole.
• A phenomenon in which a structure when exposed
to a shorter wavelength of light.
• It absorbers the energy & release it after a short time
period in the form of longer wavelength of light.
• This is known as stoke’s shift.
PRINCIPLE CONTINUED…
• The DNA sequences are identified by fluorescent
labeled probes.
• The probe is initially fluorescent labeled & then
denatured.
• The DNA of the chromosomes are also denatured.
• This is followed by hybridization of the probe & DNA.
• Counter staining is performed to observe the cells or
chromosomes.
PRINCIPLE CONTINUED…
• The stained slide are observe using the required
filter.
• Fluorechrome have characteristic color of absorption
& emission.
• A probe refers to a sequence of nucleotide which is
complementation to a gene of interest & is
sufficiently long & hybridizes uniquely to it.
 PROBE
• Probe means “To Investigate, To Search”

• A DNA (or RNA) probe is any piece of DNA (or RNA)

which has been labeled in some way and used in
hybridization assay to identify other DNA or RNA
sequences which are closely related to it in base
sequence.

• In molecular genetics, probe is utilized to investigate or

recognize complementary sequences which allows
identification and isolation of specific DNA sequence
from an organism.
 TYPES OF FISH PROBES
• Centromere
• Telomere
• Whole chromosome paint
• locus
 FISH PROBES
Gene/locus specific probes
• used to detect the presence absence or location of a
particular gene, both in metaphase and Interphase cells

Centromeres` probes
• designed to hybridize to alpha satellite repeat regions in
centromere, fluorescen brightly due to large number of
repeats in centromere.
• useful for determining the number of copies of a particular
chromosome
 TYPES OF PROBES
Whole chromosome paint probes
• made from flow-sorted or micro-dissected chromosomes
• used to determine composition of marker chromosomes,
confirm the presence of chromosome rearrangements

• Telomeric probes
have specificity for a single human chromosome arm. They
contain a locus estimated to be within 300 kb of the end of
the chromosome.
 TYPES OF PROBES

• LSI Locus Specific Identifiers

– Deletion Probes
– Translocation Probes
– Gene Detection & Localization
– Gene Amplification Probes
 FISH PROCEDURE
• Slides preparation from cultured (Metaphase) or
uncultured (Interphase) cells and fixation using
standard cytogenetic procedure.
• Denature the DNA or chromosomes
• Denature the probe
• Hybridization
• Post Washing Of Excess Probes
• Fluorescence staining
• Examine slides or store in the dark
 VISUALIZATION OF THE PROBE

• DNA probe is labeled with a colored fluorescent

molecule.
• This fluorescent molecule remains attached to the
DNA during the hybridization process
• The molecule emits a particular color when viewed
through a fluorescence microscope that is equipped
with the appropriate filter sets.
 FISH STEP by STEP
• Section cutting at Department of Histopathology.
 FISH STEP by STEP
• Slide Review and targeted area marking at
Department of Histopathology.
 FISH STEP by STEP
• Baking at 56°C
• Deparafinizing slides
• Slide pretreatment(80°C) and washing
• Protease treatment(37°C) and washing
 FISH STEP by STEP
• Dehydration
• Applying probe and cover slip on the target
area and sealing
 FISH STEP by STEP
• De-naturation (75°C for 5 min) and hybridization (37°C
for 16 hrs)
• Remove cover slip in post-hybridization buffer at room
temperature
• Post-hybridization (72°C for 2 minutes)
• Counter staining (DAPI)
• Apply cover slips, seal and freeze (-40°C)
 FISH STEP by STEP

Analyze the slide

under fluorescence
microscope that is
equipped with the
appropriate filter sets.
 FISH STEP by STEP

MICROSCOPY
 HER2:CEP17 ratio<1.8 non-amplified

RESULT: NON-AMPLIFIED HER-2 GENE

The HER2: CEP17 Ratio is less than 1.8
 FISH STEP by STEP
 HER2:CEP17 ratio 1.8-2.2 equivocal

RESULT: EQUIVOCAL HER-2 GENE

The HER2: CEP17 Ratio is 2.1
 FISH STEP by STEP
 HER2:CEP17 ratio>2.2 amplified

RESULT: AMPLIFIED HER-2 GENE

The HER2: CEP17 Ratio is greater than 2.2
FLOURESCENT MICROSCOPE
Q1.FISH technique is based on Principles of:
a) Free radicals
b) Primers
c) Fluorescent probes
d) Non of the above
e) All of the above
Q2.We use FISH technique to rule out:
f) Chromosome Micro deletion detection
g) Interphase Chromosome Enumeration
h) Gene rearrangement
i) Gene amplification
j) All of the above
Q3.Following are the probes use in FISH technique except:
k) Locus specific probes
l) Centromeric probes
m) Telomeric probes
n) Only a and b
o) C and d
Q4.For Microscopy of FISH Slides we use:
p) Simple Microscope
q) Light Microscope
r) Fluorescent Microscope
s) All of the above
t) Non of the above
Q5.Following are the important steps in FISH Procedure:
u) Denaturation of probe and target DNA
v) Hybridization of probe on complementary target sequence
w) Fluorescent Counter Staining
x) All of the above
y) Only a and b