FLUORESENT IN SITU HYBRIDIZATION

FISH (fluorescent in situ hybridization) is a cytogenetictechnique developed by biomedical

researchers in theearly 1980, which is used to detect and localize thepresence or absence of
specific DNA sequences onchromosomes. FISH uses fluorescent probes that bindto only
those parts of the chromosome with which they show a high degree of sequence
complementarity.Fluorescence microscopy can be used to find out wherethe fluorescent
probe is bound to the chromosomes.FISH is often used for finding specific features in
DNAfor use in genetic counseling, medicine, and speciesidentification.FISH can also be used
todetect andlocalize specific RNA targets (mRNA, lncRNAandmiRNA) in cells, circulating
tumor cells, and tissue samples.In this context, it can help define the spatialtemporalpatterns
of gene expression within cells and tissues.
Probes and samples used for FISH:
As mentioned earlier,FISH uses fluorescently labeled probes for the visualization of DNA
sequences

on

metaphasespreads

or

interphase

nuclei.

Both

numerical

and

structuralaberrations can be determined. Probes can be for the wholechromosome,

centromere, or locus specific. FISH probes forthe entire genome are also often used.
Interphase nucleican be obtained from a range of clinical specimens includingtouch
preparations, fine needle aspirates, bone marrowsmears, and archival material (1).
Different steps of FISH
Step 1: DenaturationConversion of double stranded DNA into single strandedform
Denaturation of labeled probe DNA
Denaturation of target DNA
(Interphase nuclei or metaphases on slide)
Step 2: Hybridization
Binding of probe DNA to target DNA
Application of probe DNA to slide and overnight incubationat 37C
Step 3: Post hybridization washing and detectionVisualization of interphase nuclei /
metaphases with boundprobe
Washing of unbound probe DNA, application of counterstainand visualization using
fluorescence microscopycells exist in clonal patches or as isolated events and toobserve
aberrations on a cell-to-cell basis rather than as a population.
Variations of FISH:

Interphase FISH
One application of FISH involves the hybridization of probesto interphase cells. This is
extremely beneficial when it isnot possible to prepare metaphase spreads as in the caseof
primary tumors. In addition, interphase FISH can beperformed on paraffin-embedded,
formalin-fixed tissuesections thereby allowing researchers to retrospectivelyanalyze samples
and correlate chromosome aberrations withbiological and clinical endpoints. Interphase
cytogeneticsalso allows one to precisely define the cell pool carryingchromosomal
abnormalities, to identify whether aberrant
Telomeric FISH (Q-FISH)
Subtelomeric probes are a relatively new addition tothe arsenal of cytogenetic tests. This test
is a collection of41 different FISH probes that are used to identifyrearrangements that cannot
be seen by conventional cytogenetic methods. The subtelomeric probes targetthe regions right
behind the ends of the chromosomesthat enables to visualise if they are involved
inrearrangements. Each of the probes is a different color sothat the specific chromosomal
segment can be identified.This is a very expensive and labor-intensive process.However, it
can be used when a geneticist suspects achromosomal abnormality and routine chromosomes
arenormal (4) or when there is chromosomal material ofunknown origin.
RNA In Situ Hybridization (RISH)
In many situations, transcription of genes at the cellularlevel needs to be studied. Several
groups have developedmethods for FISH of RNA. This is a potentially importantapplication
of the FISH technique because it provides directvisual evidence of gene expression from a
particularchromosome. Housekeeping genes, which are abundantlyexpressed, can be detected
reliably. Further optimizationand amplification of the signal can even allow detection ofgenes
expressed at baseline levels.
Primed In Situ Labelling (PRINS)
The efficacy of FISH may be limited in specificapplications by low-resolution sensitivity.
The primed insitu labelling (PRINS) method is an alternative to in situhybridization for
chromosomal detection based on the useof chromosome-specific oligonucleotide primers. In
thisprocedure, chromosomal identification is done by the insitu annealing of specific
oligonucleotide primers, followedby primer extension by TaqDNA polymerase in the
presenceof labeled nucleotides. It has been demonstrated that thePRINS technique is more
specific and considerably fasterthan classical FISH for chromosomal identification
Fiber FISH (Dynamic Molecular Combing)

The term Fiber FISH refers to the common practice offluorescence in situ hybridization
(FISH) conducted onpreparations of extended chromatin fibers. FISH on DNAfibers is useful
in assessing the length of DNA probes, andto map probes relative to one another, as it can
reveal eventheir degree of overlap. Thus, Fiber FISH has superiormapping resolution
compared to interphase FISH. It canresolve DNA loci separated by a few kilobases and
studyloci as large as two megabases in a single experiment.
Comparative Genomic Hybridization (CGH)
Comparative Genomic Hybridization serves as animportant global screening test for
chromosomal aberrationspresent within a tumor genome. This technique requiresonly
genomic tumor DNA and metaphase preps from anormal donor, thus circumventing the
preparation of highqualitytumor metaphase spreads. Tumor DNA extractedfrom archived,
formalin-fixed paraffin-embedded tissue can also be used. This allows identification of
chromosomalaberrations and facilitates the correlation of cytogeneticfindings with
histologic / histochemical information, clinicalcourse, and prognosis. Analysis of small
subregions of ahistologically defined lesion is also possible. Onceregions of gain or loss have
been identified, these regionscan be defined further using FISH or molecular
genetictechniques. CGH coupled with microarray also knownas array CGH has proved to be
very informative in many clinical settings.
Combinatorial Binary Ratio Labeling (COBRA) FISH
Combinatorial fluorescence in situ hybridization(COBRA FISH) of the DNA of the 24
different humanchromosomes with 5 fluorophores in conjunction withspectral or filter-based
microscopic

imaging

has

greatlyadvanced

molecular

cytogenetic

analysis

of

chromosomes.Use of 5 fluorophores allows the identification of up to 31different

chromosomal targets on the basis of color combinations. COBRAFISH allows
colordiscrimination of all of the p and q arms of each chromosomeand permits detection and
elucidation of intra and interchromosomal rearrangements.
Spectral karyotyping (SKY) FISH
Spectral karyotyping (SKY) is a molecular cytogenetictechnique that allows differential
visualization of all humanchromosomes in distinct colors with a single hybridization and
image exposure. After the chromosomes areclassified and aligned in a karyotype table,
interpretationand comparison of all aberrations is summarized in the karyogram.
Multiplex-FISH (M-FISH)
The M-FISH technology has the ability to identify the twentyfourdifferent human
chromosomes in a metaphase spreadby the simultaneous hybridization of chromosome-

specificDNA probes, each labeled with a different combination offluorescent dyes. M-FISH
differs from SKY only in that it isa filter-based system where separate images are
acquiredsequentially for each fluorochrome used. The individualfluorochrome files are then
combined to generate the final image.These techniques M-FISH and SKY FISH, have
combinedthe advantages of FISH with traditional chromosome bandingtechniques and
spawned many variations resulting in diverseapplications. They permit the detection of
interchromosomal structural aberrations, such as translocations and insertionsresulting in
balanced as well as unbalanced rearrangements.SKY and M-FISH have the potential to
identify

cryptictranslocations

and

clarify

complex

aberrations

(marker

andring

chromosomes), which are typically unidentifiable byconventional banding techniques. In

addition, otheraberrations such as double minutes can be better resolved, leading to the
identification of critical oncogenes.
Applications of FISH
FISH is generally used either to complement classicstaining methods or a substitute for
chromosomeidentification at metaphase or interphase. FISH has proveduseful in several
clinical settings to determine prognosis.Discrete information is obtained for each cell, which
is animportant advantage of the technique. In particular FISHdemonstrates the qualities listed
below for various diagnosticand/or prognostic applications:
Sensitivity: FISH can detect cryptic chromosomal deletionsand rearrangements, not
detectable by conventional means:submicroscopic microdeletions at the chromosomal
leveland DNA level. FISH also helps in detection ofsingle gene disorders - Duchennes
muscular dystrophy and microduplications - mental retardation.
Specificity: By using a particular probe or probes,chromosomal material of unknown or
uncertain origin canbe identified - marker chromosomes and chromosomalvariants or
polymorphisms.
Efficiency: FISH allows rapid screening of a large number ofmetaphases or interphases for a
particular chromosome orother target sequence. Mosaicism and chromosomalaneuploidies in
prenatal samples can be studied. FISHhas also proved invaluable in monitoring residual
diseasestatus in patients with cancer.
Applicability:
FISH allows interphase cells to be screenedfrom a wide variety of tissues not directly
accessible withconventional cytogenetics. Some of the studied samples are:human lung
carcinoma tissue, endometrial tissue and uncultured chorionic villus samples. FISH may
alsobe applied to buccal smear samples where venous bloodis unavailable for cytogenetic

analysis, or to blood smears, where an extremely rapid result is required. The FISHtechnique
has also provided a great deal of information about chromosome behaviour at meiosis. FISH
allows normal andabnormal chromosomes to be tracked through all stages ofmeiosis Rapid,
direct analysis of large numbers of thechromosomal complements of sperm, has been
successfullyperformed using FISH by Chantot-Bastaraudet al.Thus, FISH related applications
allow information tobe mined irrespective of whether a single locus needs to bestudied or the
entire genome needs to be scanned. Thefollowing section gives details on the impact of FISH
oncertain disciplines.
Cytogenetics
Classic cytogenetics has evolved from black and white to technicolor images of
chromosomes as a result of advancesin fluorescence in situ hybridization (FISH) techniques,
andis now called molecular cytogenetics. Improvements in thequality and diversity of probes
suitable for FISH, coupledwith advances in computerized image analysis, now permitthe
genome or tissue of interest to be analyzed in detail ona glass slide. It is evident that the
growing list of options forcytogenetic analysis has improved the understanding
ofchromosomal changes in disease initiation, progression, andresponse to treatment. The
contributions of classic andmolecular cytogenetics provided scientists and cliniciansalike
with

new

avenues

for

investigation.Small,

submicroscopic,

genomic

deletions

andduplications (1 kb to 10 Mb) constitute up to 15% of allmutations underlying human

monogenic

diseases.

Novelgenomic

technologies

such

as

microarray-based

comparativegenomic hybridization (array CGH) allow the mapping ofgenomic copy number
alterations at this submicroscopiclevel, thereby directly linking disease phenotypes to
genedosage alterations. At present, the entire human genomecan be scanned for deletions and
duplications at over 30,000loci simultaneously by array CGH (approximately 100
kbresolution), thus entailing an attractive gene discoveryapproach for monogenic conditions,
in particular those thatare associated with reproductive lethality. Fluorescence In Situ
Hybridization (FISH) showed threesignals for chromosome X (green) and two signals
forchromosome 18 (blue) confirming the karyotype results ofa structural anomaly - dicentric
X.
Prenatal Diagnosis
Prenatal diagnosis employs a variety of techniques todetermine the health and condition of an
unborn fetus.Numerical chromosome abnormalities are the major causeof inherited diseases
with an incidence of 21% inspontaneous abortions. Of these, trisomies for sexchromosomes
and chromosomes 13, 16, 18 and 21 accountfor 50% of chromosomally abnormal abortions.

Prenatal diagnosis needs a rapid, accurate and overallgenome analysis. FISH is a powerful
tool

for

detecting

somegenetic

diseases

as

well

as

microscopic

or

submicroscopicchromosome rearrangements in metaphases cells orinterphase nuclei

involving chromosomes commonlyimplicated in aneuploidies - 13, 18, 21, X and
Y.Interphase FISH is very useful in urgent high-risk cases. Theuse of FISH overcomes the
difficulties of conventional
banding on metaphase spreads. The ability to generateaccurate results in a few hours with
FISH as compared tothe two weeks typically needed for standard karyotype analysishas been
instrumental in relieving the anxiety of manywomen, or in allowing them, their families and
theirphysicians to make difficult decisions more swiftly. It isusually employed as an
adjunctive tool to conventional cytogenetics.
Fluorescence In Situ Hybridization (FISH) forchromosomes 13 and 21 showed two signals
forchromosome 21 (orange) and two signals for chromosome13 (green) indicating no
numerical anomalies associatedwith chromosomes 13 and 21.
Preimplantation Genetics
Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation
embryos prior to embryo transfer. As mentioned earlier, the correlationbetween aneuploidy
and

declining

implantation

rates

withmaternal

age

demands

screening

of

chromosomeaneuploidies in human embryos by FISH using 13, 18, 21,X and Y probes
should significantly reduce the risk of olderpatients undergoing in vitro fertilization (IVF)
delivering trisomic offspring. PGD is being explored through polarbody biopsy, biopsy of the
single cell from the eight-cell embryo, and trophectoderm biopsy of the blastocyst. Interphase
FISH using multi color, subtelomeric and centromeric probes is used to test single cells for
structuralor numerical chromosome abnormalities. It helps inidentifying embryos free of
specific genetic abnormalities. Results with PGD indicate a significant decrease
inspontaneous abortions, from 81% before PGD to 13% after PGD.Fluorescence In Situ
Hybridization (FISH) forchromosomes 13 and 21 showed two signals for chromosome 21
(orange) and one signal for chromosome13 (green) indicating that the blastomere biospied
from the embryo was abnormal.
Cancer Genetics
Chromosomal aberrations are found in cancer and tumorcell lines. Some of them are already
characterized andcorrelated with specific syndromes and some of them haveyet to be
associated with a clinical outcome. Cytogeneticanalysis is now a routine part of the diagnosis
andmanagement of a significant number of malignancies. Whilstconventional cytogenetics

remains the most comprehensive method for assessing chromosome abnormalities,

thetechnical difficulties associated with conventional cytogenetics in most cancers has
resulted in increased useof FISH to identify specific abnormalities that are useful ineither the
diagnosis or management of these disorders.Aberrations such as aneuploidies, translocations,
deletions,and gene amplifications are investigated in samples. This isaccomplished using
probes

for

centromeres,

wholechromosome

probes,

and/or

probes

for

specific

aberrantsequences.In chronic lymphocytic leukemia (CLL), geneticanalyses by FISH and

DNA sequencing have greatly improvedthe understanding of pathogenic events and
prognosticmarkers. The combination of metaphase and interphaseanalyses and the
investigation of specific structuralaberrations by FISH have definitely made tumor
diagnosticsmuch rapid and accuratein a patient referred to the Department of Human
Genetics for cytogenetic investigation. Fluorescence In Situ Hybridization (FISH) of
interphase cellsshowed a fusion signal (yellow) confirming the presence ofPhiladelphia
chromosome in a patient referred for chronicmyeloid leukemia (CML) with green / red
signalsrepresenting normal chromosomes 22 / 9.
FISH - past, present and future
Although the basic principles of FISH have remainedunchanged, high sensitivity detection,
simultaneous assayof multiple species, and automated data collection andanalysis have
advanced the field significantly. Efficiency andsensitivity have been improved by combining
methodologies.In the future, this technique is likely to have significantfurther impact on livecell imaging and on medicaldiagnostics.