Fluorescence in situ hybridization
(FISH) and the various types of FISH.
Régen DROUIN, Geneticist
MD, PhD, FACMG, FCCMG
Department of Medical Genetics, CHUS
& Department de Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Cytogenetics:
- Chromosome Cytogenetics
- Interphase Cytogenetics
- Conventional Cytogenetics
- Molecular Cytogenetics
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Molecular Cytogenetic
Techniques available:
- FISH (Fluorescence In Situ Hybridization)
& variants: Q-FISH, express FISH, etc.
- PRINS (PRimed IN Situ labeling)
- M-FISH (Multicolor-FISH) or SKY
(spectral Karyotype)
- Band-FISH
- CGH (Comparative Genomic Hybridization)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Applications of Molecular cytogenetics
 Chromosome Identification
 Aneuploidy Detection
 Centromere Analysis
 Identification of Marker Chromosome
 Whole Chromosome Analysis (chromosome painting)
 Analysis of chromosome translocation
 Detection of unique sequence (single-copy sequence)
 Microdeletion investigation
 Analysis of gene amplification
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
FISH
Fluorescence in situ hybridization
Hybridation in situ avec
visualisation en fluorescence
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
GCAATCGCCAATTATTCCAGGACTGGG
CGTTAGCGGTTAATAAGCTCCTGACCC
Double-strand DNA
Denatured DNA
Single-strand DNA
Hybridization
Fluorescence In Situ Hybridization
and Molecular Cytogenetics
1. Introduction
History:
ISH: John (1969), radioisotope probes hybridized to cell preparations
and using autoradiography to detect the hybridization of the probes.
FISH: Pinkel (1986), Immunofluorescence technique - safety, rapidity,
low background… Afterwards, so many techniques derived from FISH have
been developed, e.g. CGH, SKY, M-FISH, fiber- FISH, PRINS…
Application: Gene mapping, detecting chromosome and gene changes…
Metaphase chromosomes---from all types of cytogenetic preparations;
Interphase cells---from above plus cytomorphological preparations;
Tissue sections---from tissue biopsy slides
Principles: See Fig.
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
2. Fluorescence and fluorescence microscope
Electromagnetic spectrum: UV (<400 nm, invisible)
violet, blue, green,
yellow, orange and red (400-700 nm, visible)
infrared (>700 nm, invisible).
Energy increase with the wave length decrease.
Fluorescence:
Some fluorochromes (dyes)
FITC (Fluorescein isothiocyanate)
Rhodamine
Texas red
DAPI (4’6-diamidino-2-phenylindole)
PI (Propidium Iodide)
Fluorescence,
Long wave light
UV or short
wave light
heat
electron
Excitation (absorb energy)
Illumination (release)
Fig.: The principle of fluorochromes
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Fluorescence microscope:
Light source: High-pressure mercury vapor lamps, tungsten-halogen lamps, or
Eye or Camera
xenon lamps.
Filters: 1. Exciting filter, to let a certain
wave length of light pass so that
can excite the given fluorochrome
carried on sample.
2. Barrier filter, to allow the visible
light pass so that the fluorescence
can be seen by eyes or the image
can be captured.
Transmitted and epi-illumination
Special requirements: No auto-fluorescence
in any part of light path except for
samples
Specimen on microscope slide
3. Probes---a specific DNA fragment, usually 1 to 100 kb
length, complementary to the chromosome site
that we are interested in.
Probe Labeling: a). Indirect labeling, need antibodies to complete FISH procedure
Haptens---Biotin-dUTP, digoxigenin-dUTP,
b). Direct labeling, the probe directly labeled with fluorochromes
such as SpectralGreen and SpectralOrange.
One-step hybridization.
Labeling techniques: a). Nick translation
b). Random priming
c). PCR (Polymerase chain reaction)
DNase I makes nicks
DNA Polymerase adds dNTP, labeled dUTP at 3’ and remove dNTP at 5’
5‘
3‘
5‘
3‘
5‘
3‘
+※++※+
+※++※
Fig.: Principle of Nick Translation
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
++※+++
3‘
5‘
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
4. Hybridization
a). Denaturing of DNA probes
b). Denaturing of DNA template (chromosome)
c). Annealing (renature, hybridization)
d). Post-hybridization wash, stringency control
Factors
Level
Stringency
Results
(if inappropriate)
Temperature
High
Low
High
Low
Low efficiency
High background
Concentration of salt
(SSC) solution
High
Low
Low
High
High background
Low efficiency
Concentration of
formamide solution
High
Low
High
Low
Low efficiency
High background
e). Counterstaining
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
4. Hybridization
a). Denaturing of DNA probes
b). Denaturing of DNA template (chromosome)
c). Annealing (renature, hybridization)
d). Post-hybridization wash, stringency control
e). Counterstaining
Probe color(s)
Counterstain should be used
FITC (Green)
PI (Red)
Rhodamine or Texas Red (Red)
DAPI (Blue)
Mixed probe with multicolor
DAPI (Blue)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
FISH targets :
-
Metaphase Chromosomes
Interphase Nuclei
Fixed Tissues
Cells in culture
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Extended section
of chromosome
Chromatin fiber of
packed nucleosomes‘String-of-beads’ DNA double helix
form of chromatin
Condensed section
of chromosome
10nm
700nm
30nm
300nm
1 400nm
2 nm
DNA
Condensation
and fiberFISH
A good FISH method
should have:
- An extremely high specificity
(extremely low background)
- A good sensitivity (good
hybridization efficiency)
- Unambiguous recognition of the
hybridization signal
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Genetic diseases identified using
molecular cytogenetics
•
•
•
•
•
•
•
•
•
Prader-Willi Syndrome
Angelman Syndrome
Miller-Dieker Syndrome
Williams Syndrome de Williams
DiGeorge and velo-cardio-facial Syndromes
Wolf-Hirschhorn Syndrome
Smith-Magenis Syndrome
Kallmann Syndrome
etc...
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Di George
normal
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Di George
normal
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Deletion
of
one
Di George
locus
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Prader-Willi Syndrome
(del.15q11-q13 pat ou DUP mat)
 Mental Retardation and behavior problems
 Deglutition problems and hypotonic newborn
 Bulimia presented by older children (obesity)
 Hypogonadism and incomplete puberty
 Acromicry
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Angelman Syndrome
(del.15q11-q13 mat ou DUP pat)
 Severe Mental Retardation
 Episodes of uncontrolled laughing
 Characteristic Facial Dysmorphism (low jaw and protruding tongue)
 Special behavior with disorganized movements (ataxic gait)
 Convulsions
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
PWS
SNRPN
15q11q13
15q22
control
PML
15q22
control
PML
SNRPN
15q11q13
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
PWS
15q22
control
PML
del (15) (q11q13)
15q22
control
PML
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
SNRPN
15q11q13
DEFINITION OF CRYPTIC
CHROMOSOME REARRANGEMENTS
These are chromosomal anomalies not visible using
standard high resolution cytogenetic technique ( 
550 bands per haploid genome). These anomalies
are detectable only when using molecular
cytogenetic techniques.
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Pedigree
Normal
Normal
Miller-Dieker Syndrome
Deceased
MDS
Department of Medical Genetics, CHUS &
Department
of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Deceased MDS ?
Current pregnancy (MDS)
Spontaneous Abortion
Father
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Father
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
FISH (Oncor)
MDS Probes 17p13.3 et RARA CR 17q21.1
16
17
17
Father of the propositus
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
PRINS
PRimed IN Situ
labeling
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Telomere
Simple DNA sequence (T2AG3) tandemly repeated, of variable
length, located at the extremities of the chromosomes.
Telomeres are essential elements that protect the extremities of
the chromosomes from degradation and ligation.
Shortening
Incomplete Replication
Nuclease Activity
Senescence
Equilibrium
Elongation
Addition of repetitions
T2AG3 by the telomerase
Telomeres
• Specialized structures made of DNA and PROTEINS
• Repeated DNA sequence: 2 à 15 kb
• Maintain the chromosome stability
TTAGGG TTAGGG TTAGGG
AATCCC AATCCC AATCCC
• Around 30 to 120 bp are lost per somatic cell division
• Too short : cellular senescence and genetic instability
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Measurement of telomeres
Average length of telomeres :
• Measurement of terminal restriction fragments.
– Digestion using restriction enzymes of purified DNA
– Visualization and measurements of telomeric fragments by
Southern blot
• Cleavage of telomeres at variable distance
• No information individual telomeres
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Measurement of telomeres
Length of individual telomeres :
Quantitative FISH (Q-FISH)
Hybridization telomeric PNA probes
Measurements of the signal intensity
Length Profil of individual telomeres
Variation of hybridization efficiency ???
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
1. Measurement of 1q telomere
2.9 m
100kb/29 m
100kb/19.5 m
100kb/14.1 m
2.1 m
1.5 m
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
A
B
C
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
The multi-color Karyotype
This is a technique that allows simultaneous
identification and analysis of all chromosomes by
attributing to each pair of autosomes, to the X and Y
chromosomes, a specific and distinct color.
There are 2 methods:
Multiplex-Fluorescent In Situ Hybridization (M-FISH)
(Speicher et al., 1996)
SKY (Spectral Karyotyping)
(Schröck et al., 1996)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
M-FISH
Sequential Acquisition of images corresponding to
each fluorochrome, using 5 optic filters specific for
the different fluorochromes.
FITC
TexasRed
DEAC
15
9
X
9
SpectrumOrange
Cy 5
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
SKY
The main part of the system: an
optic head composed of an
interferometer that measures the
fluorescence spectrum and a CCD
camera for the imaging.
The emission spectrum of the
fluorescence can be
simultaneously recorded for
each point of the image.
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Cell lines from a colorectal adenocarcinoma
3
12
3
X
del(X)
ins(3;12)
4
del(4)
8
19
17
iso(13q)
der(18)
der(19)t(17;19)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
der(8)
5
5
6
6
3
1
9
2
13
14
1
der(1)t(1;5)
B6-15
X
9
X
der(2)t(1;2)
der(3)t(X;3)
B6-1, B6-15
HT29, C7-1, C7-15
der(5)t(5;13)
der(6)t(6;14)
HT29, C7-1, C7-15
12
HT29, C7-1, C7-15
der(6)t(X;6;9)
B6-1, B6-15
5
15
9
9
X
X
9
9
13
7
14
6
ins(X;9)
der(9)t(X;6;9)
der(12)t(7;12)
der(13)t(5;13)
der(14)t(6;14)
HT29
C7-15
B6-15
HT29, C7-1
HT29, C7-1, C7-15
dup(19p)
C7-1, C7-15
Representative Karyotype of the cell line
del(3p)
der(6)t(6;14)
del(Xp)
der(8)
del(7p)
ins(9;X)
iso(13q)
der(13)t(5;13)
der(14)t(6;14)
13
der(5)t(5;13)
del(4)
14
der(18)
15
16
17
18
dup(19p)
der(19)t(17;19)
19
20
21
22
m-FISH Karyotype
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
m-FISH Karyotype (False Colors)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Chromosome Painting
Painting 19 red & 8 green
Painting 19 red & 5 green
19
19
19
8
19
?
?
19
5
8
19
5
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
m-FISH
inv(2)(q23;q34)
der(1)t(1;9)(q21;q12)
der(2)t(2;12)(q35;q12)
del(3)(q10)
i(3)(q10)
der(7)t(7;13)(q31;q12)
der(5)t(5;20)(q15;p12)
der(7)t(inv7;14)[(q21;q34)(q22;q24)]
der(9)t(1;9)(q21;q12) der(10)t(3;10;12)(3qter3q21::12?::10p1510q26::10q2410qter)
der(9)t(8;9)(q12;p13)
i(12)(p11)
der(14)t(6;14)(q11.1;p11.1)
der(19)t(5;8;19)(19pter19q13.1::8?::19q13.219q13.3::5q345qter)
der(19)t(8;19)(?;p11)
del(18)(?)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
der(20)t(5;20)(q15;p12)
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Comparative Genomic Hybridization
protocols
Preparation of Metaphases
• Preparation of
Genomic DNA
• DNA labeling
Preparations
• Hybridization
• DNA detection
• Image Capture
• Image Treatment
• Image Analysis
Normal
Genomic DNA
Genomic DNA of
the tumor
Hybridization
Metaphase
Preparations
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada
Team of Dr Régen DROUIN
Cytogenetics
Molecular Genetics
• Walid DRIDI
Macoura GADJI
Kada KRABCHI
Josée LAVOIE
• Sandrine LACOSTE
Stéphane OUELLET
Patrick ROCHETTE
François VIGNEAULT
• Éric BOUCHARD
Marc BRONSARD
• Ju YAN
• Nathalie BASTIEN
Mélissa FERLAND
Isabelle PARADIS
Department of Medical Genetics, CHUS & Department of Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada