FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s
that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
FISH uses fluorescent probes that bind to only those parts of the chromosome with which
they show a high degree of sequence complementarity. 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 in cells, circulating
tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns
of gene expression within cells and tissues.
Probes – RNA and DNA
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. This technique
[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 bit/s. 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. The size of the human genome is so large, compared
to the length that could be sequenced directly, that it was necessary to divide the genome
into fragments. To preserve the fragments with their individual DNA sequences, the fragments
were added into a system of continually replicating bacteria populations. Clonal populations of
bacteria, each population maintaining a single artificial chromosome, are stored in various
laboratories around the world. The artificial chromosomes can be grown, extracted, and labeled,
in any lab. These fragments are on the order of 100 thousand base-pairs, and are the basis
for most FISH probes. Preparation and hybridization process – RNA
Cells, circulating tumor cells, or formalin-fixed paraffin-embedded or frozen tissue sections
are fixed, 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). Separate but compatible signal amplification systems enable the multiplex
assay. Signal amplification is achieved via a series of sequential hybridization steps.
At the end of the assay the tissue samples are visualized under a fluorescence microscope.
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 PCR 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. FISH experiments designed to detect or localize
gene expression within cells and tissues rely on the use of a reporter gene, such as one
expressing green fluorescent protein, to provide the fluorescence signal.
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; and how they are used in combination.
Probes are divided into two generic categories: cellular and acellular."In situ" in Fluorescence
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
peptides. These few modifications make possible all FISH techniques.
Probe size is important because longer probes hybridize less specifically than shorter probes,
"a short strand of DNA or RNA which is complementary to a given target sequence, it 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 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.
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,
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 of DNA; these mixtures are used to detect
deletion mutations. When combined with a specific colour, 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 unique enough to identify each chromosome A variety of other techniques use mixtures
of differently colored probes. A range of colors in mixtures of fluorescent dyes can
be detected, so each human chromosome can 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 colour is observed, but only
the primary colours are observed when the translocation occurs. This technique is sometimes
called "break-apart FISH". 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, fresh, or frozen, and in samples
of cells and circulating tumor cells. ViewRNA ISH probes allow single molecule RNA sensitivity
with virtually no background. Each probe is composed of 20 oligonucleotide pairs. Each
oligo pair forms a required platform for assembly of the signal amplification structure through
a series of sequential hybridization steps using the branched DNA signal amplification
technology. Each fully assembled structure, covers a space of 40–50 bit/s of the target
mRNA/lncRNA, and has the capacity for 400-fold signal amplification. Therefore, a typical
target specific probe has the capacity to generate 8,000-fold signal amplification.
Independent but compatible signal amplification systems enable simultaneous detection of up
to four target mRNAs/lncRNAs in cells/CTCs and up to two mRNAs/lncRNAs in tissues.
Stellaris(R) 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 labeled oligonucleotide probes. The binding of up to 48 fluorescent
labeled oligos to a single molecule of mRNA provides sufficient fluorescence to accurately
detect and localize each target mRNA in a wide-field fluorescent microscopy image. Probes
not binding to the intended sequence do not achieve sufficient localized fluorescence
to be distinguished from background. Single-molecule RNA FISH assays can be performed
in simplex or multiplex, and can be used as a follow-up experiment to quantitative PCR,
or imaged simultaneously with a fluorescent antibody assay. The technology has potential
applications in cancer diagnosis, neuroscience, gene expression analysis, and companion diagnostics.
Fiber FISH In an alternative technique to interphase
or metaphase preparations, fiber FISH, 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 shear along the length of the slide, either to cells
that have been fixed to the slide and then lysed, or to a solution of purified DNA. A
technique known as chromosome combing is increasingly used for this purpose. The extended conformation
of the chromosomes allows dramatically higher resolution - even down to a few kilobases.
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-FISH combines FISH with PNAs and computer
software to quantify fluorescence intensity. This technique is used routinely in telomere
length research. Flow-FISH
Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence
measurements. Medical applications
Often parents of children with a developmental disability want 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 and cytogenetic techniques.
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, and Down syndrome. FISH on sperm cells is
indicated for men with an abnormal somatic or meiotic karyotype as well as those with
oligozoospermia, 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 a diagnosis,
to evaluate prognosis, or to evaluate remission of a disease, such as cancer. 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 standard Cytogenetic methods, 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-chip microfluidic 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. Species identification
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen,
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 biological species, to deduce evolutionary relationships. A similar hybridization technique
is called a zoo blot. Bacterial FISH probes are often primers for the 16s rRNA region.
FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms,
for example, are composed of complex 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 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 Hybridization Comparative genomic hybridization can 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 Karyotype
Virtual karyotyping is another cost-effective, clinically available alternative to FISH panels
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 Karyotype
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. See also
In situ hybridization, the technique used for labelling
Chromogenic in situ hybridization Molecular cytogenetics
Virtual Karyotype Happy mapping
Gallery
Notes and references Pernthaler A, Pernthaler J, Amann R. "Fluorescence
In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine
Bacteria". Applied and Environmental Microbiology 68: 3094–3101. doi:10.1128/AEM.68.6.3094-3101.2002.
PMC 123953. PMID 12039771. Wagner M, Horn M, Daims H. "Fluorescence in
situ hybridisation for the identification and characterisation of prokaryotes". Current
Opinion in Microbiology 2003: 302–309. doi:10.1016/S1369-5274(03)00054-7.
External links Fluorescence in Situ Hybridization at the
US National Library of Medicine Medical Subject Headings
Information on fiber FISH from the Olympus Corporation
A guide to fiber FISH from Octavian Henegariu Fibre FISH protocol from the Human Genome
Project at the Sanger Centre CARD-FISH, BioMineWiki
Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes
FISH technical notes and protocols from GeneDetect.com Fluorescence in situ Hybridization Photos
of bacteria Rational design of polynucleotide probe mixes
to identify particular genes in defined taxa: www.dnaBaser.com/PolyPro