FLUORESCENCE IN SITU

HYBRIDIZATION
Dr. Dimple Mehrotra
 HISTORICAL ASPECTS
The classic cytogenetic staining was the result of dyes that bind
to the DNA or protein of a chromosome and allow visualization by
light microscopy.

In situ hybridization, a combination of molecular and cytogenetic

technologies, opened another door that has allowed further
investigation of chromosome anomalies.

Here, instead of a dye, a molecular probe (i.e., a fragment of DNA)

binds to the chromosome.

In early studies, the probe was labeled with a radioactive compound

(usually tritium) such that after 510 days of exposure to radiograph
film, a pattern of silver grains could be detected identifying the
chromosomal location of that probe. This technique facilitated gene
mapping by identifying gene loci.
 Autoradiography was useful, but only one probe at a time
could be used; it required a significant length of time for
exposure, and there was a degree of scatter of the silver grains
that necessitated statistical analysis of the data obtained.

In the mid-1980s, a variation of the technique was developed

that has proven to be extremely useful in clinical applications.

The new technology, fluorescence in situ hybridization

(FISH), was quicker, more specific, and allowed the use of
multiple probes in a single hybridization procedure.

Furthermore, instead of radioactively labeled probes, a

fluorescent dye was used, that was visualized using
fluorescence microscopy
 INTRODUCTION
FISH is acytogenetictechnique used to detect and localize the presence or
absence of specificDNAsequencesonchromosomes.

FISH usesfluorescent probesthat bind to only those parts of the chromosome

with which they show a high degree of sequencecomplementarity.

Fluorescence microscopycan be used to find out where the fluorescent probe is

bound to the chromosomes.

FISH is often used for finding specific features in DNA for use in genetic
counseling, medicine, and species identification.

FISH can also be used to detect and localize specific RNA targets (mRNA
andmiRNA) in cells, circulating tumor cells, and tissue samples. In this
context, it can help define the spatial-temporal patterns of gene
expressionwithin cells and tissues.
 DEFINITION
In situ hybridization is the
method of localizing/ detecting
specific nucleotide sequences in
morphologically preserved
tissue sections or cell
preparations by hybridizing the
complementary strand of a
nucleotide probe against the
sequence of interest.

If nucleic acids are preserved

in a histological specimen, then
it can be detected by using a
complementary probe
PRINCIPLE OF FISH
Principle is same as that of ISH

Use of a fluorescent labeled probe differentiates

ISH & FISH

FISH is a cytogenetic technique used to detect

and localize the presence or absence of specific
DNA sequences on chromosomes
PROTOCOL OUTLINE
TYPES OF PROBES USED
In clinical applications of FISH, the most common goal is to
determine whether
a gene,
a specific mutation, or
a particular chromosomal rearrangement ,is present

So the molecular probes used must be well characterized and

specific to the locus in question.

There are three basic types of probes:-

1. Chromosome painting probes
2. Repeat sequence probes
3. Unique sequence probe
I) Chromosome painting probes are actually a cocktail of many
unique DNA fragments from along the entire length of a chromosome,
such that, following hybridization, the entire chromosome fluoresces .

Whole chromosome paint probe highlighting the two X chromosomes in a female

cell
II)Repeat sequence probes are isolated from
1. telomere or
2. centromere regions

. Centromere probes are usually used in chromosome enumeration

(i.e., to detect the gain or loss of specific chromosomes)

. Atrue telomere probe recognizes the six base repeat present at

the ends of all chromosomes and will confirm the presence or
absence of the telomeric regions.

. Inpractice, however, both true centromere and telomere probes

are not particularly useful because so many signals are
generated.

. Chromosome-specific pericentromeric and subtelomeric

probes are now in use.
C and D, Repeat sequence centromere probe for chromosome 21.

C, Trisomy 21 seen at left in an interphase nucleus and at right in a metaphase

(arrows).

D, Normal complement of two copies of chromosome 21 (arrows) seen in

interphase (left and right) and in a metaphase (center).
III) A unique sequence probe is usually isolated from:-
1. cloned DNA of a disease-causing gene or
2. a fragment of DNA of known location associated with a
particular gene.

This type of probe is used to identify the presence or absence of

the :-
3. gene,
4. gene region, or
5. chromosomal rearrangement of interest

Subtelomere probes are a subset of this category.

. Research revealed that there are unique sequences just

proximal to the telomere of each chromosome arm
that can be used for specific identification of each arm.
Chromosomes showing hybridization with subtelomere probes.
The p arm subtelomere is indicated by the green signals, and the q arm
subtelomere is indicated by the red signals.
Paired red and green signals are seen in the interphase nucleus to the left.
 The subtelomere probes that have been generated
have become very valuable in characterizing cryptic
rearrangements by determining whether all
subtelomeres are present and located on the correct
chromosome arms, and, if not, if a rearrangement
has occurred.

Furthermore, it has been shown that 3%5% of

patients with unexplained mental retardation have a
cytogenetically undetectable terminal deletion of a
chromosome.

Subtelomere FISH now allows identification of such

deletions and localization to the specific chromosome
affected
TECHNIQUE
FISH can be performed on either metaphase or
interphase cells.

For metaphase FISH, cells are cultured and harvested

as for a routine karyotype analysis, but no culture is
required for interphase FISH.

Fixed cells are collected and slides are made

The DNA on the slides is then denatured, and a

fluorescently labeled single-stranded molecular probe
is allowed to hybridize to the chromosomal DNA using
an annealing temperature that favors hybridization of
homologous regions of DNA .
Schematic of fluorescence in situ hybridization (FISH) technology.
DNA derived from a known gene is rendered single stranded, labeled
with a fluorescent tag, then hybridized back to the chromosomes of a
metaphase cell.
 After an appropriate period of hybridization, excess probe is
washed away and the nonhybridized DNA is counterstained with
another fluorochrome to allow visualization of the entire
chromosome complement

A fluorescent microscope with light from a 100 W mercury bulb

in combination with appropriate sets of exciter filters allows
evaluation of the cells.

Due to the optical limitations of the glass filters, a maximum of

three colors can be visualized on a typical fluorescent microscope.

For documentation, a 35-mm camera attached to the microscope

can be used, although superior results are obtained using a
computer assisted system to acquire the fluorescent images.

Software packages also allow enhancement of weak signals and

sharpening of the image.
 One of the most critical elements in FISH is the selection of a
specific probe or probes that will help to answer a clinical question.

For example, one of the most useful clinical applications of FISH is

the detection of microdeletions too small to be seen using
classical cytogenetics

The gene must be known and the probe used must be homologous
to the critical region of the gene that is usually deleted.

Hybridization with the probe should result in a fluorescent signal

only at the target locus. If a signal is present, DNA complementary
to the probe is present, so there is no deletion
Depiction of a metaphase cell with 46
chromosomes that has been processed using
FISH technology.
The labeled probes hybridized to targets are
indicated with arrows.
Dual signals indicating hybridization to both
alleles of target gene
 However, the absence of signal
indicates that a deletion exists
(i.e., there is no DNA sequence
present on the chromosome that
is complementary to the probe,
so no hybridization can occur)

Hybridization of both controls (green) but presence of only a single target gene
indicating deletion of one allele.

A control probe to a different

region of the chromosome being
tested is usually used as a
hybridization control.

Target gene indicated by red signal and a control probe indicated in green.
Complete hybridization to all alleles
Therefore, when evaluating a disease related probe,
An Unaffected individual should have two signals per cell for each
autosomal gene, one signal for each chromosome of the pair
An Affected individual with a deletion should have a single signal
per cell, showing one normal and one deleted chromosome
For Both affected and unaffected, the control probe should show two
signals per cell in all cells that are scored.
 In a metaphase cell, the chromosomes may be short and
condensed, with each chromatid individually visible,
resulting in a discrete FISH signal on each chromatid.

In these cells, there may appear to be two signals per

chromosome for a total of four signals but, in actual
scoring, only complete chromosomes are considered so a
signal on one or both chromatids would be counted as
just one positive signal.

One of the problems with this detection system is that

the lack of signal is the positive indication of a
deletion.

Technical failure of hybridization may also result in

absence of signal.
 To eliminate this as a source of error,
a minimum of 20 cells must be evaluated, and all cells
must agree in signal count.

If mosaicism is suspected, additional cells may be

surveyed.

In addition, unique sequence probes are currently used

in combination with a control probe.

The secondary probe is localized to the same arm of

the chromosome as the deletion locus.
TYPES OF SAMPLES USED
FISH PROCEDURE FOR PARAFFIN
EMBEDDED TISSUE
FISH PROCEDURE FOR FROZEN
TISSUE
CHROMOSOME PAINTING PROBES
are most useful in identifying complex rearrangements or marker
chromosomes.

If an individual has an abnormal chromosome with extra material

of unknown origin, it may be possible to use chromosome painting
to identify the source of the extra DNA.
This may help in either diagnosis or prognosis of the individual.

For example, in the case of a patient with only one identifiable X

chromosome and a small, unidentified marker chromosome, it will
be important to chromosome. determine if the origin of the marker is
an X or a Y

This can be accomplished using chromosome paints for the X and Y

chromosomes labeled with different fluorochromes
 FISH probes are most informative when evaluated in
metaphase cells , where it is possible to identify the
specific chromosome to which the probe hybridizes.

However, in some cell preparations, very few

metaphase cells may be present, and, in these cases,
information may be obtained with unique sequence or
repeat sequence probes in interphase nuclei.

Here, the technical problems are increased as a result

of random loss of signal, extra signals, and overlapping
signals in cells, so additional cells (a total of 50200)
must be counted to obtain a statistically significant
result.
 DUAL-COLOR FISH (DFISH)
employs two probes with different fluorescence wavelengths to identify
structural chromosomal rearrangements.

Each probe generates a characteristic color by itself (split signal) and a

third color when the two probes are juxtaposed (fusion signal).

A fusion signal indicating chromosomal rearrangement is used to identify

disease-causing mutations that predictably involve only two partner
genes, for example, BCR and ABL.

However, when a particular gene with multiple potential translocation

partners is tested, it is more efcient to have both probes bind to the 5
and 3 ends of the particular gene such that the normal allele shows the
fusion signal and the rearranged allele shows two split signals.

This is the technique used for the MLL gene, which is rearranged to over
30 different partner genes in various types of leukemia
MULTICOLOR FLUORESCENCE IN SITU
HYBRIDIZATION
Another advantage of FISH is the ability to hybridize
multiple probes to a single slide to obtain a better
understanding of chromosome rearrangements

For most FISH probes, the maximum number of colors is

three (one target sequence, one control sequence, and one
counterstain), and this can be handled by a typical
fluorescent microscope.

However, if more than three colors are needed, a computer-

assisted imaging system is required. This is because a
fluorescent image is the result of the light from the mercury
bulb passing through an exciter filter, hitting the
fluorochrome on the slide, and transmitting an image of the
appropriate color to the eyepiece for viewing.
 Each fluorochrome requires its own filter so, for
each additional color desired, the light must pass
through additional thicknesses of glass. The
optical properties of glass and light limit the total
number of viewable colors to three.

However, with a computer-assisted system,

detection of multiple colors becomes possible. The
computer does not actually see more colors but
it can detect subtle differences in shades better
than the human eye. In a relatively simple
example, two fluorochromes are used and mixed
in varying proportions to give a range of colors.
 In this example
a red fluorochrome is mixed with

a yellow fluorochrome

Generating intermediate colors from red to yellow.

Eight signals are seen, but as viewed through the microscope, it is impossible

to be absolutely sure which signal corresponds to a particular chromosome.

Although difcult to distinguish by the human eye, the computer can register

each color as a different shade of gray and then the software assigns a unique
color to that shade for display on the computer monitor
 Using the key to the left, the following chromosomes are detected:
two each for chromosomes 13, 21, and X;
three chromosomes 18, and
one Y chromosome.

This cell is therefore trisomic for chromosome 18 with an XXY sex

chromosome complement.

Combinations of fluorochromes have been developed that allow detection of

each of the 24 different chromosomes followed by a unique color assignment by
the computer
 The same physics that make a variety
of colors possible for M-FISH can be
used for the detection of translocations.

That is, colors that are adjacent appear

to overlap; a secondary color is
observed. Some assays are designed so
that the secondary color will be present
or absent in cases of interest.

An example is the detection ofBCR/

ABLtranslocations, where the
secondary color indicates disease.

This variation is often called double-

fusion FISH or D-FISH.
 In the opposite situationwhere the absence of
the secondary color is pathologicalis illustrated
by an assay used to investigate translocations
where only one of the breakpoints is known or
constant. Locus-specific probes are made for one
side of the breakpoint and the other intact
chromosome. In normal cells, the secondary
colour is observed, but only the primary colours
are observed when the translocation occurs. This
technique is sometimes called "break-apart
FISH"
VARIATIONS ON PROBES AND
ANALYSIS

FISH is a very general technique. The differences between

the various FISH techniques are usually due to:
variations in the sequence and labeling of the probes
how they are used in combination

Probes are divided into two generic categories:

cellular
Acellular

"In situ" in Fluorescent in situ hybridization refers to the

placement of the probe placed cellularly.

FISH is usually associated with nucleic acid based-type

methods that can be further broken down to antimicrobial
[Link] few modifications make possible all FISH
 Probe size is important because longer probes
hybridize less specifically than shorter probes,

"a short strand of DNA or RNA (often 10-25 nucleotides)

which is complementary to a given target sequence, can
be used to identify or locate the target."

The overlap defines the resolution of detectable features.

For example, if the goal of an experiment is to detect the

breakpoint of atranslocation, then the overlap of the
probes the degree to which one DNA sequence is
contained in the adjacent probes defines the minimum
window in which the breakpoint may be detected.
 The mixture of probe sequences determines the type of feature
the probe can detect.

Probes that hybridize along an entire chromosome are used to count

the number of a certain chromosome, show translocations, or identify
extra-chromosomal fragments ofchromatin. This is often called
"whole-chromosome painting.

If every possible probe is used, every chromosome, (the whole genome)

would be marked fluorescently, which would not be particularly useful
for determining features of individual sequences.

However, it is possible to create a mixture of smaller probes that

are specific to a particular region (locus) of DNA; these mixtures are
used to detectdeletion mutations.

When combined with a specific color, a locus-specific probe mixture

is used to detect very specific translocations. Special locus-specific
probe mixtures are often used to count chromosomes, by binding to
thecentromericregions of chromosomes, which are unique enough to
identify each chromosome
 RECENT ADVANCES
Spectral karyotyping (SKY) and multiplex FISH
(MFISH) are relatively new advancements of
conventional FISH that utilize multiple
fluorochromes, specialized optics, and image analysis
that can simultaneously identify all chromosomes.

Multiplex RNA visualization in cells using ViewRNA FISH Assays

 Comparative genomic hybridization (CGH), also called
copy number karyotyping
is a variation of FISH that detects relative gains or losses of the
[Link] method compares the ratios of patient specimen
DNA, labeled with one fluorochrome to that of normal DNA,
labeled with a different fluorochrome when hybridized to control
chromosomes.

CGH is used predominantly in research to identify possible

pathways involved in tumor progression, recurrence, or
metastasis.

Colorimetric probes are used in chromogenic in situ

hybridization (CISH), which has the advantage that the signal
does not fade with time or require fluorescence microscopy for
analysis.
QUANTIGENE VIEWRNA ISH PROBES
QuantiGene ViewRNA ISH probes is a method of detecting
and quantifying mRNA, lncRNA and miRNA molecules in
tissue samples that are FFPE (Formalin-fixed parafn-
embedded), fresh, or frozen, and in samples of cells
andcirculating tumor cells(CTC).

ViewRNA ISH probes allow single molecule RNA sensitivity

with virtually no background.

Each probe (for the detection of mRNA and lncRNA) is

composed of 20 oligonucleotide pairs.

Each oligo pair forms a required platform for assembly of the

signal amplification structure (tree) through a series of
sequential hybridization steps using the branched DNA
(bDNA) signal amplification technology.
STELLARIS RNA FISH PROBES
Stellaris RNA FISH, formerly known as Single Molecule RNA
FISH, is a method of detecting and quantifying mRNA and other long
RNA molecules in a thin layer of tissue sample.

Targets can be reliably imaged through the application of multiple

short singly labeledoligonucleotide probes.

The binding of up to 48fluorescent labeledoligos to a single molecule

of mRNA provides sufcient fluorescence to accurately detect and
localize each target mRNA in a wide-fieldfluorescent
microscopyimage.

Single-molecule RNA FISH assays can be performed in simplex

ormultiplex, and can be used as a follow-up experiment
toquantitative PCR, or imaged simultaneously with afluorescent
antibodyassay.

The technology has potential applications incancer diagnosis,

neuroscience, gene expressionanalysis
 FIBER FISH
In an alternative technique tointerphaseor metaphase preparations,
fiber FISH- in which- interphase chromosomes are attached to a slide

in such a way that they are stretched out in a straight line, rather than
being tightly coiled, as in conventional FISH, or adopting a random
conformation, as in interphase FISH.

This is accomplished by applying mechanical shearalong the length of

the slide,
either to cells that have been fixed to the slide and thenlysed, or
to a solution of purified DNA.

A technique known aschromosome combingis increasingly used for

this purpose.

The extended conformation of the chromosomes allows dramatically

higher resolution. The preparation of fiber FISH samples, although
conceptually simple, is a rather skilled art, and only specialized
laboratories use the technique routinely.
 Q-FISH
Q-FISHcombines FISH withPNAsand
computer software to quantify fluorescence
intensity. This technique is used routinely
intelomere length research.
 FLOW-FISH
Flow-FISHusesflow cytometryto perform FISH
automatically using per-cell fluorescence
measurements
 MEDICAL APPLICATIONS
Often parents of children with adevelopmental disabilitywant to know more
about their child's conditions before choosing to have another child. These
concerns can be addressed by analysis of the parents' and child's DNA.

In cases where the child's developmental disability is not understood, the

cause of it can potentially be determined using FISH
andcytogenetictechniques.

Examples of diseases that are diagnosed using FISH include

Prader-Willi syndrome,
Angelman syndrome,
22q13 deletion syndrome,
chronic myelogenous leukemia,acute lymphoblastic leukemia,
Cri-du-chat,
Velocardiofacial syndrome,
Down syndrome.
Interphase cells positive for a chromosomal t(9;22)
rearrangement
Urothelial cells marked with four different probes
ViewRNA detection of miR-133(green) and myogenin mRNA (red) in
C2C12 differentiating cells
 Detection of BCR/ABL in chronic myelogenous
leukemia

A metaphase cell positive for the bcr/ablrearrangement using FISH.

The chromosome that is labeled with greenandred spots (upper left) is the one where
the rearrangement is present.
 Detection of HER2 gene amplification for breast
cancer diagnosis and prognosis
 Used in the analysis of trisomies (chromosome
gains) and monosomies (chromosome losses)
associated with leukaemias and lymphomas. The
presence of trisomy is detected as three
fluorescent dots within the cell whilst monosomy
is seen as a single fluorescent dot within the cell.
 Detection of MYC amplification in neuroblastoma

FISH utilizing probes against centromere 8 (green) and c-myc (red ) shows
innumerable c-myc signals, consistent with high-level gene amplification
 FISH onsperm cellsis indicated for men with

an abnormal somatic or meiotickaryotype, and

those witholigozoospermia, (since approximately
50% of oligozoospermic men have an increased
rate of sperm chromosome abnormalities.)

The analysis of chromosomes 21, X, and Y is

enough to identify oligozoospermic individuals at
risk
 In medicine, FISH can be used to:
form adiagnosis
evaluateprognosis,
evaluateremissionof a disease, such ascancer.

Treatment can then be specifically tailored.

A traditional exam involving metaphase chromosome

analysis is often unable to identify features that distinguish
one disease from another, due to subtle chromosomal
features; FISH can elucidate these differences.

FISH can also be used to detect diseased cells more easily

than standardCytogeneticmethods, which require dividing
cells and requires labor and time-intensive manual
preparation and analysis of the slides by a technologist.
.

FISH, on the other hand, does not require living

cells and can be quantified automatically, a
computer counts the fluorescent dots present.

However, a trained technologist is required to

distinguish subtle differences in banding
patterns on bent and twisted metaphase
chromosomes.

FISH can be incorporated into

Lab-on-a-chipmicrofluidic device.
This technology is still in a developmental stage
but, like other lab on a chip methods, it may lead
to more portable diagnostic techniques

Microfluidic chip that lowered the cost per

test of FISH by 90%
SPECIES IDENTIFICATION
FISH is often used inclinical studies. If a patient is
infected with a suspectedpathogen, bacteria from the
patient's tissues or fluids, are typically grown on agar to
determine the identity of the pathogen.

Many bacteria, however, even well-known species, do not

grow well under laboratory conditions. FISH can be used to
detect directly the presence of the suspect on small samples
of patient's tissue.

FISH can also be used to compare the genomes of two

biologicalspecies, to deduceevolutionaryrelationships.

A similar hybridization technique is called a

zoo blot. Dual label FISH image;
Bifidobacteria
 FISH is widely used in the field ofmicrobial
ecology, to identifymicroorganisms.

Biofilms, for example, are composed of complex

(often) multi-species bacterial organizations.

Preparing DNA probes for one species and

performing FISH with this probe allows one to
visualize the distribution of this specific species
within the biofilm.

Preparing probes (in two different colors) for two

species allows to visualize/study co-localization of
these two species in the biofilm, and can be useful
in determining the fine architecture of the biofilm.
 Comparative genomic hybridizationcan be
described as a method that uses FISH in a parallel
manner with the comparison of the hybridization
strength to recall any major disruptions in the
duplication process of the DNA sequences in the
genome of the nucleus.
 Virtual karyotypingis another cost-effective,
clinically available method using thousands to
millions of probes on a single array to detect copy
number changes, genome-wide, at unprecedented
resolution. Currently, this type of analysis will
only detect gains and losses of chromosomal
material and will not detect balanced
rearrangements, such as translocations and
inversions which are hallmark aberrations seen
in many types of leukemia and lymphoma.
 Spectral karyotyping is an image of colored
chromosomes. Spectral karyotyping involves
FISH using multiple forms of many types of
probes with the result to see each chromosome
labeled through its metaphase stage. This type of
karyotyping is used specifically when seeking out
chromosome arrangements.
SUMMARY
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