1. FLUOROSCENT INSITUFLUOROSCENT INSITU
HYBRIDIZATIONHYBRIDIZATION
Fluorescent in situ hybridization (Fluorescent in situ hybridization (FISHFISH) is a) is a
powerful technique for detecting RNA or DNApowerful technique for detecting RNA or DNA
sequences in cells, tissues, and tumors. FISHsequences in cells, tissues, and tumors. FISH
provides a unique link among the studies ofprovides a unique link among the studies of
cell biologycell biology, cytogenetics, and molecular, cytogenetics, and molecular
geneticsgenetics.. ItIt is a technique in which single-is a technique in which single-
stranded nucleic acids (usually DNA, but RNAstranded nucleic acids (usually DNA, but RNA
may also be used) are permitted to interact somay also be used) are permitted to interact so
that complexes, or hybrids, are formed bythat complexes, or hybrids, are formed by
molecules with sufficiently similar,molecules with sufficiently similar,
complementarycomplementary sequences.sequences.

2. ByBy nucleic acidnucleic acid hybridization, the degree ofhybridization, the degree of
sequence identity can be determined, andsequence identity can be determined, and
specific sequences can be detected and locatedspecific sequences can be detected and located
on a givenon a given chromosomechromosome
The method comprises of three basic steps:The method comprises of three basic steps:
fixation of a specimen on afixation of a specimen on a microscopemicroscope slide,slide,
hybridization of labeled probe to homologoushybridization of labeled probe to homologous
fragments of genomic DNA, and enzymaticfragments of genomic DNA, and enzymatic
detection of the tagged target hybridsdetection of the tagged target hybrids

3. The probe sequences were initially detected withThe probe sequences were initially detected with
isotopic reagents, nonisotopic hybridization hasisotopic reagents, nonisotopic hybridization has
become increasingly popular, with fluorescentbecome increasingly popular, with fluorescent
hybridization now a common choice. Protocolshybridization now a common choice. Protocols
involving nonisotopic probes are considerablyinvolving nonisotopic probes are considerably
faster, with greater signal resolution, and providefaster, with greater signal resolution, and provide
options to visualize different targetsoptions to visualize different targets
simultaneously by combining various detectionsimultaneously by combining various detection
methods.methods.

4. The detection of sequences on the targetThe detection of sequences on the target
chromosomes is performed indirectly, commonlychromosomes is performed indirectly, commonly
with biotinylated or digoxigenin-labeled probeswith biotinylated or digoxigenin-labeled probes
detected via a fluorochrome-conjugateddetected via a fluorochrome-conjugated
detection reagent, such as an antibodydetection reagent, such as an antibody
conjugated with fluorescein, as a result, theconjugated with fluorescein, as a result, the
direct visualization of the relative position of thedirect visualization of the relative position of the
probes is possible.probes is possible.

5. nucleic acid probes labeled directly withnucleic acid probes labeled directly with
fluorochromes are used for the detection of largefluorochromes are used for the detection of large
target sequences. This method takes lesstarget sequences. This method takes less timetime
and results in lower background; however, lowerand results in lower background; however, lower
signal intensity is generated. Higher sensitivitysignal intensity is generated. Higher sensitivity
can be obtained by building layers of detectioncan be obtained by building layers of detection
reagents, resulting in amplification of the signal.reagents, resulting in amplification of the signal.
Using such means, it is possible to detect single-Using such means, it is possible to detect single-
copy sequences on chromosome with probescopy sequences on chromosome with probes
shorter than 0.8 kb.shorter than 0.8 kb.

6. Probes can vary in length from a few base pairsProbes can vary in length from a few base pairs
for synthetic oligonucleotides to larger than onefor synthetic oligonucleotides to larger than one
Mbp. Probes of different types can be used toMbp. Probes of different types can be used to
detect distinct DNA types. PCR-amplifieddetect distinct DNA types. PCR-amplified
repeated DNA sequences, oligonucleotidesrepeated DNA sequences, oligonucleotides
specific for repeat elements, or cloned repeatspecific for repeat elements, or cloned repeat
elements can be used to detect clusters ofelements can be used to detect clusters of
repetitive DNA in heterochromatin blocks orrepetitive DNA in heterochromatin blocks or
centromeric regions of individual chromosomescentromeric regions of individual chromosomes

7. FISH is useful in determining aberrations in theFISH is useful in determining aberrations in the
number of chromosomes present in a cell. Innumber of chromosomes present in a cell. In
contrast, for detecting singlecontrast, for detecting single locuslocus targets,targets,
cDNAs or pieces of cloned genomic DNA, fromcDNAs or pieces of cloned genomic DNA, from
100bp to 1Mbp in size, can be used. To detect100bp to 1Mbp in size, can be used. To detect
specific chromosomes or chromosomal regions,specific chromosomes or chromosomal regions,
chromosome-specific DNA libraries can be usedchromosome-specific DNA libraries can be used
as probes to delineate individual chromosomesas probes to delineate individual chromosomes
from the full chromosomal complement Specificfrom the full chromosomal complement Specific
probes have been commercially available forprobes have been commercially available for
each of the human chromosomes since 1991.each of the human chromosomes since 1991.

8. A givenA given tissuetissue or cell source, such as sections ofor cell source, such as sections of
frozen tumors, imprinted cells, cultured cells, orfrozen tumors, imprinted cells, cultured cells, or
embedded sections, may be hybridized. Theembedded sections, may be hybridized. The
DNA probes are hybridized to chromosomesDNA probes are hybridized to chromosomes
from dividing (metaphase) or non-dividingfrom dividing (metaphase) or non-dividing
(interphase) cells. The observation of the(interphase) cells. The observation of the
hybridized sequences is done usinghybridized sequences is done using
epifluorescenceepifluorescence microscopymicroscopy. White. White lightlight from afrom a
source lamp is filtered so that only the relevantsource lamp is filtered so that only the relevant
wavelengths for excitation of the fluorescentwavelengths for excitation of the fluorescent
molecules reach themolecules reach the samplesample or non-dividingor non-dividing
(interphase) cells.(interphase) cells.

9. The light emitted by fluorochromes is generallyThe light emitted by fluorochromes is generally
of larger wavelengths, which allows theof larger wavelengths, which allows the
distinction between excitation anddistinction between excitation and emissionemission
light by means of a second opticallight by means of a second optical
filter.Therefore, it is possible to see brightfilter.Therefore, it is possible to see bright
colored signals on a dark background. It is alsocolored signals on a dark background. It is also
possible to distinguish between severalpossible to distinguish between several
excitation and emission bands, thus betweenexcitation and emission bands, thus between
several fluorochromes, which allows theseveral fluorochromes, which allows the
observation of many different probes on theobservation of many different probes on the
same target.same target.

10. APPLICATIONAPPLICATION
FISH has a large number of applications inFISH has a large number of applications in
molecular biologymolecular biology and medical science,and medical science,
includingincluding genegene mapping,mapping, diagnosisdiagnosis ofof
chromosomal abnormalitieschromosomal abnormalities, and studies of, and studies of
cellular structure and function. Chromosomes incellular structure and function. Chromosomes in
three-dimensionally preserved nuclei can bethree-dimensionally preserved nuclei can be
"painted" using FISH. In clinical research, FISH"painted" using FISH. In clinical research, FISH
can be used for prenatal diagnosis of inheritedcan be used for prenatal diagnosis of inherited
chromosomal aberrations, postnatal diagnosis ofchromosomal aberrations, postnatal diagnosis of
carriers of geneticcarriers of genetic diseasedisease, diagnosis of, diagnosis of
infectious disease, viral and bacterial disease,infectious disease, viral and bacterial disease,
tumor,tumor, cytogenetic diagnosis, and detection ofcytogenetic diagnosis, and detection of
aberrant gene expression .aberrant gene expression .

11. In laboratory research, FISH can be used forIn laboratory research, FISH can be used for
mapping chromosomal genes, to study themapping chromosomal genes, to study the
evolutionevolution of genomes (Zoo FISH), analyzingof genomes (Zoo FISH), analyzing
nuclear organization, visualization ofnuclear organization, visualization of
chromosomal territories andchromosomal territories and chromatinchromatin inin
interphase cells, to analyze dynamic nuclearinterphase cells, to analyze dynamic nuclear
processes, somaticprocesses, somatic hybridhybrid cells, replication,cells, replication,
chromosome sorting, and to study tumorchromosome sorting, and to study tumor
biologybiology ..It can also be used in developmentalIt can also be used in developmental
biology to study the temporal expression ofbiology to study the temporal expression of
genes during differentiation and development.genes during differentiation and development.
Recently, high resolution FISH has become aRecently, high resolution FISH has become a
popular method for ordering genes or DNApopular method for ordering genes or DNA
markers within chromosomal regions of interestmarkers within chromosomal regions of interest