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.

ApplicationsFISH 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.
Structural Abnormalities: 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 can visualize 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

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 of these 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 Nmyc, 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

cosmids, bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), yeast artificial chromosomes (YACs), chromosome band or arm specific sequences generated by microdissection, and DNA libraries established by chromosome flow sorting. To make the probes, DNA sequences are labeled directly with fluorescent dyes or indirectly with biotin or digoxigenin, which are then detected with immunofluorescent staining. DNA sequences can be labeled with a single color, dual colors, or multiple colors. Single or multiple probes can be used for each hybridization. In recent years, numerous fluorescent labeled DNA probes have been commercially available, making the FISH protocols much simpler, more costeffective, and reliable for clinical applications.

Labeling methods
FISH requires nucleic acid probes, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or nucleic acid analogs, labelled directly with fluorophores, or capable of indirect association with fluorophores. The nucleic acid provides the FISH assay with its specificity through complementary pairing of the probe nucleotides with nucleotides of the target nucleic acid. The appended fluorophores provide the ability to visually detect the homologous regions within the cellular structure using a fluorescence microscope. Photographic or electronic cameras can also be used to provide permanent images of the fluorescence staining patterns, and the latter can be used to provide quantitative measurements of the probe fluorescence.

1) Direct Labeling
Fluorophores can be associated with nucleic acid probes by chemical conjugation to the nucleic acid. This method is called as direct labelling One or more fluorescent dye molecules are directly bound to the oligonucleotide either chemically during synthesis through an amino linker at the 5-end of the probe or enzymatically using terminal transferase to attach fluorescently labelled nucleotides at the 3-end. It is by far the most common, cheapest and easiest way of labelling FISH probes. Large probes can be easily labelled using the direct method. Although this method does not require the need of additional incubation steps the disadvantage of this method is the signal intensity cannot be amplified.

2) Indirect Labelling
Fluorophores can be associated with nucleic acid probes by chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization. This method is called indirect labelling.

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, the indirect 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 doublestranded fragments must be denatured prior to hybridization.

Fig 7: DNA labeling by Nick Translation.

Fig 8: DNA labelling by Random Priming

Random Priming-Random priming is a means of labelling DNA fragments whereby, a

mixture of all possible combinations of hexamers, octamers, or nonamers are annealed to denatured DNA. These small oligonucleotides then act as primers that allow for synthesis of the complementary DNA strand by the Klenow enzyme and incorporation of both labelled and unlabeled nucleotides. The labelled material will be a combination of both double- and single stranded fragments that must be denatured prior to hybridization

DOP-PCR- PCR generated whole chromosome DNA, either from microdissected or flow
sorted chromosomes, is currently viewed as the preferred route to high quality probes suitable for whole chromosome staining of individual chromosomes. This is true for staining single chromosomes as well as for staining all 24 human chromosomes combinatorially in a multiplex FISH (M-FISH) or spectral karyotyping assay. The reaction involves the use of an oligonucleotide with an Xho-I restriction endonuclease site at its 5' end, a defined six-nucleotide sequence at the 3' end, and a set of degenerate nucleotides (a random mix of all 4 nucleotides) in-between.

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.

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 also useful 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 used to 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 single chromosome (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.

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)