Lecture Notes (M.Sc.

Biotechnology, IInd Semester)

Fluorescence in situ Hybridization

1. Introduction:

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that

uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a
high degree of sequence complementarity.
It was developed by biomedical researchers in the early 1980s to detect and localize the
presence or absence of specific DNA sequences on chromosomes.
Fluorescence microscopy can 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, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples.

2. Types of Probes:
A probe is a single strand of DNA or RNA that is complementary to a nucleotide
sequence of interest.
RNA probes can be designed for any gene or any sequence within a gene for
visualization of mRNA, lncRNA and miRNA in tissues and cells.
FISH is used by examining the cellular reproduction cycle, specifically interphase of the
nuclei for any chromosomal abnormalities.
FISH allows the analysis of a large series of archival cases much easier to identify the
pinpointed chromosome by creating a probe with an artificial chromosomal foundation
that will attract similar chromosomes.
The hybridization signals for each probe when a nucleic abnormality is detected.
Each probe for the detection of mRNA and lncRNA is composed of 20 oligonucleotide
pairs, each pair covering a space of 40-50 bp.
For miRNA detection, the probes use proprietary chemistry for specific detection of
miRNA and cover the entire miRNA sequence.
Probes are often derived from fragments of DNA that were isolated, purified, and
amplified for use in the Human Genome Project.

3. Preparation and Hybridization Process – RNA:

Cells, circulating tumor cells (CTCs), or formalin-fixed paraffin-embedded (FFPE) or

frozen tissue sections are fixed, and then permeabilized to allow target accessibility.
FISH has also been successfully done on unfixed cells. A target-specific probe, composed
of 20 oligonucleotide pairs, hybridizes to the target RNA(s).

Page 1
 Lecture Notes (M.Sc. Biotechnology, IInd Semester)

Separate but compatible signal amplification systems enable the multiplex assay. Signal
amplification is achieved via series of sequential hybridization steps.
At the end of the assay the tissue samples are visualized under a fluorescence
microscope.

4. Preparation and Hybridization Process – DNA:

First, a probe is constructed. The probe must be large enough to hybridize specifically
with its target but not so large as to impede the hybridization process.
The probe is tagged directly with fluorophores, with targets for antibodies or with biotin.
Tagging can be done in various ways, such as nick translation, or Polymerase chain
reaction using tagged nucleotides.
Then, an interphase or metaphase chromosome preparation is produced. The
chromosomes are firmly attached to a substrate, usually glass.
Repetitive DNA sequences must be blocked by adding short fragments of DNA to the
sample. The probe is then applied to the chromosome DNA and incubated for
approximately 12 hours while hybridizing.
Several wash steps remove all unhybridized or partially hybridized probes. The results
are then visualized and quantified using a microscope that is capable of exciting the dye
and recording images.
If the fluorescent signal is weak, amplification of the signal may be necessary in order to
exceed the detection threshold of the microscope.
Fluorescent signal strength depends on many factors such as probe labeling efficiency,
the type of probe, and the type of dye.
Fluorescently tagged antibodies or streptavidin are bound to the dye molecule. These
secondary components are selected so that they have a strong signal.

5. Variation on Probe and Analysis:

The differences between the various FISH techniques are usually due to variations in the
sequence and labeling of the probes; and how they are used in combination.
Probes are divided into two generic categories: cellular and acellular. In fluorescent "in
situ" hybridization refers to the cellular placement of the probe.
Probe size is important because longer probes hybridize less specifically than shorter
probes, so that short strands of DNA or RNA (often 10-25 nucleotides) which are
complementary to a given target sequence are often used to locate a target.
The overlap defines the resolution of detectable features. For example, if the goal of an
experiment is to detect the breakpoint of a translocation, 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.

Page 2
 Lecture Notes (M.Sc. Biotechnology, IInd Semester)

Fig. 01: FISH experiments to localize a gene in the nucleus

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
of chromatin. 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 detect deletion 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 the centromeric regions of chromosomes, which are distinctive enough to identify each
chromosome (with the exception of Chromosome 13, 14, 21, 22.)
A variety of other techniques uses mixtures of differently colored probes. A range of
colors in mixtures of fluorescent dyes can be detected, so each human chromosome can

Page 3
 Lecture Notes (M.Sc. Biotechnology, IInd Semester)

be identified by a characteristic color using whole-chromosome probe mixtures and a

variety of ratios of colors.
Although there are more chromosomes than easily distinguishable fluorescent dye colors,
ratios of probe mixtures can be used to create secondary colors.
Similar to comparative genomic hybridization, the probe mixture for the secondary colors
is created by mixing the correct ratio of two sets of differently colored probes for the
same chromosome. This technique is sometimes called M-FISH.
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 of BCR/ABL translocations, where the secondary
color indicates disease.
This variation is often called double-fusion FISH or D-FISH. In the opposite situation—
where the absence of the secondary color is pathological is 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 color is observed, but only the primary
colors are observed when the translocation occurs. This technique is sometimes called
"break-apart FISH".

6. Applications:
Species Identification
Comparative Genomic Hybridization
Virtual Karyotype
Spectral Karyotype
Disease Diagnosis by FISH

7. References:

Gerald Karp - Cell and molecular biology. 5th Edition (2007).

Lewis J. Klein smith and Valerie M. Kish - Principles of cell and molecular biology -
Third Edition (2002).
Richard M. Twyman-Advanced Molecular Biology, First South Asian 'Edition." (1998),
Viva Books Pvt. Ltd.
Benjamin Lewin, Gene IX, 9th EditionJones and Barlett Publishers, 2007.
J.D. Watson, N.H. Hopkins, J.W Roberts, J. A. Seitz.& A.M. Weiner; Molecular Biology
of the Gene, 6th Edition, Benjamin Cummings Publishing Company Inc, 2007.
TA Brown - Genomes 2nd Edition; Bios Scientific Publishers 2002 Harvey Lodish,

Page 4
 Lecture Notes (M.Sc. Biotechnology, IInd Semester)

Arnold Berk, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, Anthony Bretscher,
Hidde Ploegh and Paul Matsudaira - Molecular Cell Biology, 6h Edition; WH Freeman
2008.

Langer-Safer, P. R.; Levine, M.; Ward, D. C. (1982). "Immunological method for

mapping genes on Drosophila polytene chromosomes". Proceedings of the National
Academy of Sciences. 79 (14): 4381-5.
Amann, Rudolf; Fuchs, Bernhard M. (2008). "Single-cell identification in microbial
communities by improved fluorescent in situ hybridization techniques". Nature Reviews
Microbiology. 6 (5): 339-348.
Anthony, S. J.; St. Leger, J. A.; Pugliares, K.; Ip, H. S.; Chan, J. M.; Carpenter, Z. W.;
Navarrete-Macias, I.; Sanchez-Leon, M.; Saliki, J. T.; Pedersen, J.; Karesh, W.; Daszak,
P.; Rabadan, R.; Rowles, T.; Lipkin, W. I. (2012). "Emergence of Fatal Avian Influenza
in New England Harbor Seals". mBio. 3 (4): e00166-e00112.
Everitt, A. R.; Clare, S.; Pertel, T.; John, S. P.; Wash, R. S.; Smith, S. E.; Chin, C. R.;
Feeley, E. M.; Sims, J. S.; Adams, D. J.; Wise, H. M.; Kane, L.; Goulding, D.; Digard, P.;
Anttila, V.; Baillie, J. K.; Walsh, T. S.; Hume, D. A.; Palotie, A.; Xue, Y.; Colonna, V.;
Tyler-Smith, C.; Dunning, J.; Gordon, S. B.; Everingham, K.; Dawson, H.; Hope, D.;
Ramsay, P.; Walsh (Local Lead Investigator), T. S.; et al. (2012). "IFITM3 restricts the
morbidity and mortality associated with influenza". Nature. 484 (7395): 519-23.
Louzada, S.; Adega, F.; Chaves, R. (2012). "Defining the sister rat mammary tumor cell
lines HH-16 cl.2/1 and HH-16.cl.4 as an in vitro cell model for Erbb2". PLOS One. 7 (1):
e29923.
Ting, D. T.; Lipson, D.; Paul, S.; Brannigan, B. W.; Akhavanfard, S.; Coffman, E. J.;
Contino, G.; Deshpande, V.; Iafrate, A. J.; Letovsky, S.; Rivera, M. N.; Bardeesy, N.;
Maheswaran, S.; Haber, D. A. (2011). "Aberrant Overexpression of Satellite Repeats in
Pancreatic and Other Epithelial Cancers". Science. 331 (6017): 593-6.
Zhang, B.; Arun, G.; Mao, Y. S.; Lazar, Z.; Hung, G.; Bhattacharjee, G.; Xiao, X.; Booth,
C. J.; Wu, J.; Zhang, C.; Spector, D. L. (2012). "The lncRNA Malat1 is Dispensable for
Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult". Cell
Reports. 2 (1): 111-23.
Lee, K.; Kunkeaw, N.; Jeon, S. H.; Lee, I.; Johnson, B. H.; Kang, G. -Y.; Bang, J. Y.;
Park, H. S.; Leelayuwat, C.; Lee, Y. S. (2011). "Precursor miR-886, a novel noncoding
RNA repressed in cancer, associates with PKR and modulates its activity". RNA. 17 (6):
1076-89.
Bernasconi, B.; Karamitopolou-Diamantiis, E.; Tornillo, L.; Lugli, A.; Di Vizio, D.;
Dirnhofer, S.; Wengmann, S.; Glatz-Krieger, K.; Fend, F.; Capella, C.; Insabato, L.;
Terracciano, L. M. (2008). "Chromosomal instability in gastric mucosa-associated
lymphoid tissue lymphomas: A fluorescent in situ hybridization study using a tissue
microarray approach". Human Pathology. 39 (4): 536-42.
Haroon, Mohamed F.; Skennerton, Connor T.; Steen, Jason A.; Lachner, Nancy;
Hugenholtz, Philip & Tyson, Gene W. (2013). "Chapter One - In-Solution Fluorescent In
Situ Hybridization and Fluorescence-Activated Cell Sorting for Single Cell and
Population Genome Recovery". In F. DeLong Edward (ed.). Methods in
Enzymology. 531. Academic Press. p. 3-19.

Page 5