fish - no, not that one

8/13/2019 FISH - No, not that one

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FISH: The technique and its clinical

applications

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting and mapping the

position of DNA and RNA sequences in cells, tissues, and tumors.

It enables the localization of specific DNA sequences to interphase chromatin and metaphase

chromosomes. It is sensitive, versatile and most extensively used cytochemical staining

technique.

Technique and its applications

FISH for visualization of nucleic acids developed as an alternative to older methods that used

radio labeled probes. Early methods of isotopic detection employed non-specific labeling

strategies, such as the random incorporation of radioactive modified bases into growing cells,

followed by autoradiography.

FISH allows significant advances in resolution, speed and safety, and has paved the way for the

development of simultaneous detection of multiple targets, quantitative analyses and live-cell

imaging.

The first application of fluorescent in situ detection came in 1980, when RNA that was directly

labeled on the 3

end with fluorophore was used as a probe for specific DNA sequences.

Applications-

Structural Abnormalities: FISH analysis is necessary for the identification and

characterization of most unbalanced de novo structural rearrangements, including marker

chromosomes. Numerous acquired aberrations which lead to gains or losses of chromosomal

material have been described in leukemia lymphomas, and solid tumors.

Microdeletion Syndromes: Williams, Prader-Willi/Angelman, Smith-Magenis, 22q11.2

deletion, and 1p36 deletion, are the most common microdeletion syndromes. Routine banding

technique including high resolution banding can cover a region up to 850 bands, which canvisualize deletions as small as 2 to 5 Mb. But anything smaller, needs a technique with a higher

resolution and for most microdeletion syndromes a definitive diagnosis cannot be made

without FISH analysis.

Detection of Subtelomeric Aberrations in Patients with Unexplained Mental

Retardation: It was suggested that subtelomeric anomalies may be second only to Down


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syndrome as the most common cause of mental retardation. Patients with unexplained mental

retardation or developmental disabilities have been studied by FISH with multiple subtelomeric

probes.

Prenatal Diagnosis of the common Anueploidies: Aneuploidies of chromosomes 13, 18,

21, X, and Y account for about 95% of the chromosomal aberrations causing live-born birth

defects. This technique is particularly valuable for high risk pregnancies as indicated by

ultrasonography or maternal serum screening.

Prenatal Diagnosis of Chromosomal Disorders using maternal blood: Fetal nucleated

red blood cells which pass into the maternal circulation during pregnancy provide a cell source

for noninvasive prenatal genetic diagnosis. Cytogenetic analysis of fetal cells by FISH is a

potentially useful method for prenatal diagnosis of chromosomal disorders, but requires

relatively pure samples of fetal cells isolated from maternal blood.

Preimplantation Diagnosis of the common Aneuploidies:Since most aneuploidies arise as

the products of a maternal meiosis I non-disjunction, they can be detected by FISH analysis on

the first and/or second polar bodies removed from oocytes following maturation and

fertilization. DNA probes for chromosomes 13, 18, and 21 have been used most commonly for

FISH studies on polar bodies.

Detection of Specific Translocations and Gene Rearrangements in human Cancer:

Over 100 recurrent chromosomal translocations in hematologic neoplasms, malignant

lymphomas, and solid tumors have been identified, and rearrangement of a specific gene is

known in most of these translocations. FISH has been a powerful tool in the characterization ofthese translocations. It has been shown that interphase FISH is highly sensitive in detecting the

BCR/ABL fusion, and therefore is very useful for following patients response to therapy.

Testing Deletion of Tumor Suppressor Genes and amplification of Oncogenes:Deletion

of tumor suppressor genes, such as p53 and RB-1, and amplification of oncogenes, such as N-

myc, C-myc, and HER-2/neu, can be detected by FISH. FISH has provided reliable estimates of

N-myc amplification in neuroblastoma. While the changes in copy number of multiple

oncogenes can be simultaneously tested using CGH microarrays, a large number of tumor tissue

specimens can be rapidly analyzed with FISH performed on consecutive tissue microarray

sections.

Sources of Probe DNA

All types of human DNA sequences have been used as probes for molecular cytogenetic studies.

These include unique sequences, repetitive sequences such as -satellite and telomere DNA,

locus specific DNA obtained by PCR amplification, large genomic DNA sequences cloned into


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In indirect labelling, the molecule directly attached to the nucleic acid probe is typically

either biotin or a hapten, such as dinitrophenol (DNP) or digoxigenin.

The in situ hybridization is performed with the hapten- or biotin-labelled probe, after

which the specimen is incubated with fluorophore-labelled antibody or avidin. Because

a number of fluorophores can be attached to each antibody or avidin molecule, theindirect method allows for the association of multiple fluorophores with each directly

attached binding moiety.

Furthermore, additional rounds of antibody binding, sometimes referred to as

sandwiching, can be utilized to further increase the number of bound fluorophores.

While indirect labelling has the potential for generating greater fluorescence signal, it

also has the disadvantage of requiring additional incubation steps to bind the antibody

and avidin reagents.

The introduction of fluorescent antibodies also can increase the background

fluorescence owing to nonspecific binding of the antibodies and avidin proteins to

extraneous cellular material on the microscope slide, and the slide surface itself.

Furthermore, when multicolor FISH is utilized to simultaneously identify several

different genomic targets, a different, spectrally distinct fluorophore must be used to

unambiguously identify each of the targets.

Nick translation-Nick translation is a method for incorporating labelled nucleotides into

DNA such as an isolated fragment or an intact clone.

The method uses a combination of two enzymes, deoxyribonuclease I (DNase I)which nicks the

DNA creating free 3' hydroxyls, and DNA polymerase I, which processively adds nucleotides to

the 3' terminal hydroxyl.

The 5' to 3' exonuclease activity of the DNA polymerase removes nucleotides from the 5'

terminus of the nick as the polymerization proceeds.

Both labelled and unlabeled nucleotides are substituted during the reaction and varying sized

fragments are generated; however, there is no net synthesis of DNA. The resultant double-

stranded fragments must be denatured prior to hybridization.


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Flourophores for FISH

Table 2: The figure shows a list of flurophores, their extinction coefficients, excitation and

emission wavelengths. (Morrison LE., et.al, 2002)

Gene-specific probes

Gene-specific probes target specific nucleic acid sequences within chromosomes. Examples of

such probes include bacterial artificial chromosome (BAC) and yeast artificial chromosome

(YAC) probes and cosmids. These probes have proven particularly useful in the study of

microdeletion syndromes, where the absence of a gene often goes undetected by conventional

banding methods.


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For instance, Duchenne muscular dystrophy is a progressive muscular degenerative disease that

results from a deletion at Xp21 (i.e. band 21 on the short arm of the X chromosome) in affected

males. Although the deletion can vary in size, exon 45 of the dystrophin gene is missing in 60%

of patients. FISH probes that can detect deleted regions of this gene are elegant tools for

determining the carrier status of clinically unaffected women. Gene-specific probes are alsouseful for mapping genes on chromosomes.

Repetitive-sequence probes

Repetitive-sequence probes bind to regions that are rich in repetitive base-pair sequences.

Examples of such probes include centromeric and telomeric probes. Centromeres frequently

contain AT-rich tandem repeats, whereas telomeres are recognized by the short repetitive

sequence TTAGGG. Centromeric probes have applications in the identification of marker

chromosomes and numerical chromosome abnormalities in interphase nuclei and when

specimens are sex mismatched. Telomeric probes and subtelomere specific are commonly usedto identify cryptic chromosomal translocations such as those occurring in cases of unknown

mental retardation.

Whole-genomic DNA probes

Whole-genomic DNA probes are used for the FISH-based technique CGH. They can be used to

detect genomic imbalances in tumor genomes by combining tumor and normal DNA to analyze

gains and losses.

Chromosome-painting probes

Chromosome-painting probes contain sequences that are specific to either a singlechromosome (i.e. whole-chromosome-painting probes) or an arm of a chromosome (i.e.

chromosome arm- painting probes).

After hybridization, one or more chromosomes of interest are lit up in different colours, which

are dependent on which particular fluorochromes have been used. This technique is particularly

useful for identifying chromosome arms that are involved in translocations, as well as for

marker chromosomes and ring chromosomes. Ring chromosomes are formed when breaks

occur in the short and long arms of a chromosome, and the broken ends re-join to form a ring.

Whole-chromosome-painting probes are also used in SKY. By assigning an individual colour to

each of the 23 pairs of human chromosomes, an entire karyotype (i.e. all 46 chromosomes) can

be differentially labelled. As well as being useful for visualizing all aberrations in a simple

experiment, SKY can be used to elucidate cryptic translocations that often go undetected by

conventional cytogenetic analysis.


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Figure 4: Different types of probes ((N. McNeil and T. Ried, 2000)

Spectral karyotyping (SKY)is a technique that combines the power of conventional

chromosome analysis with the specificity of FISH.

SKY can be used to identify marker chromosomes (i.e. chromosomes that are important in the

diagnosis of a disease) and detect telomeric translocations, which are sometimes difficult to

identify using traditional banding analysis. This technique has also proven to be beneficial in

elucidating the complex rearrangements that are observed in cancer genomes. SKY entails a

single multicolour FISH analysis, which can be used to yield 24 different-coloured chromosomes

in a human metaphase spread. SKY involves a combination of epifluorescence microscopy,

charge-coupled device (CCD) imaging and

Fourier spectroscopy to measure the

complete emission spectra at all image

points.

Figure 3: Procedure of spectral karyotyping

((N. McNeil and T. Ried, 2000)

fish - no, not that one

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