Fluorescence in Situ Hybridization (FISH) Test

Introduction

The most reliable method for identifying specific DNA sequences, diagnosing genetic disorders, mapping genes, and identifying novel oncogenes or genetic abnormalities causing different kinds of cancers is fluorescence in situ hybridization or FISH. In fluorescence inverted microscope (FISH) analysis, target sequences of the sample DNA are annealed to fluorescent reporter molecules using DNA or RNA probes.

Recently, the technique has been extended to allow simultaneous screening of the entire genome using multicolor whole chromosome probe techniques like spectral karyotyping or multiplex FISH or via an array-based approach utilizing comparative genomic hybridization. In the fight against genetic illnesses, FISH is today acknowledged as a dependable diagnostic and discovery technique, having radically transformed the field of cytogenetics.

What Are the Principles of FISH?

The fundamentals of in situ fluorescence hybridization.

• A DNA probe and a target sequence are the fundamental components.

• The DNA probe is either directly labeled by including a fluorophore or indirectly labeled with a hapten before hybridization.

• Single-stranded DNA is produced by denaturing the target DNA and the labeled probe.

• After that, they are joined, causing complementary DNA sequences to anneal.

• If the probe has been indirectly labeled, an additional procedure that employs an enzymatic or immunological detection technique is needed to see the nonfluorescent hapten. Finally, fluorescence microscopy is used to assess the signals.

What Is the Role of Probes in FISH?

Selecting the right probe is one of the most crucial aspects of a FISH examination. One can utilize a broad variety of probes, ranging from modest cloned probes (1 to 10 kb) to entire genomes. There are generally three sorts of probes: whole chromosome painting probes, repeating sequence probes, and locus-specific probes, each with a distinct set of uses. These are briefly discussed below.

• Chromosome Painting: A process known as "chromosome painting" involves hybridizing fluorescently labeled composite probe pools specific to individual chromosomes to cytological preparations. This makes chromosomal abnormalities visible and makes it possible to see individual chromosomes in cells that are in metaphase or interphase.

• Short sequences that are present in thousands of copies are found in certain chromosomal regions or structures that repetitive sequence probes hybridize to. Pan-telomeric probes, for instance,

  • Specifically, target the tandemly repeated (TTAGGG) sequences found at the ends of every human chromosome.

  • The α- and β-satellite sequences, which flank the centromeres of human chromosomes, are the target of centromeric probes.

  • In both metaphase and interphase diploid cells, satellite DNA probes hybridize to multiple copies of the repetitive sequences found in the centromeres, producing two incredibly brilliant fluorescent signals.

  • These centromere-specific probes help identify various aneuploidies, such as trisomy and monosomy, in solid tumors and leukemias.

• Genomic clones, which are typically locus-specific probes, come in different sizes based on the type of cloning vector used. These include plasmids, which can carry 1–10 kb; PAC (P1 bacteriophage-derived artificial chromosome, which can carry 100–300 kb); YAC (yeast artificial chromosome, which can carry 150–350 kb); and RAC vectors, which can carry 80 kb to 1 Mb. In both metaphase and interphase, these probes are especially helpful for detecting translocations, inversions, and deletions.

What Are the Steps Involved in In Situ Hybridization?

• Cytological Preparation: For optimal morphology and maximum hybridization signals, preparation should be well-distributed and flat. Mitotic root tip preparations have been used in the majority of ISH research on plant chromosomes. After being preserved in ethanol or glacial acetic acid, the root tips are crushed in 45 percent acetic acid and dyed with one percent acetocarmine on the slide. Slides can be kept for a minimum of a year in a freezer at -80 degrees Celsius. Before hybridization, the chromosomes are dried out on the slide after they have thawed.

• Probe Labeling: Several techniques have been developed for labeling DNA probes for nonradioactive in situ hybridization. The most popular method involves using reporter molecules (haptens) to label the probe. There are numerous haptens on the market, including coumarin, AMCA, fluorescein, digoxigenin, biotin, and dinitrophenol. These haptens can be integrated as labeled nucleotides using random primer labeling, nick translation, or PCR tagging techniques following standard protocols. Anti-digoxigenin antibodies coupled to enzymes or fluorochromes facilitate the detection of hybridized digoxigenin probes. The spin column or ethanol precipitation techniques can be used to separate the labeled DNA from unincorporated nucleotides. The random primed labeling technique works by hybridizing a mixture of all conceivable hex nucleotides with the target DNA.

• In Situ Hybridization:

  • Nonradioactive in situ hybridization.

  • Fluorescence in situ hybridization (FISH).

  • Genomic in situ hybridization (GISH).

What Uses Does FISH Serve?

• Locus-specific probes bind a chromosome's specific area. Scientists can employ this kind of probe when they have isolated a little bit of a gene and wish to find out which chromosome the gene is located on or how many copies of a gene are present in a given genome.

• Locus-specific probes bind a specific area of a chromosome. When researchers have isolated a tiny gene segment and wish to identify the chromosome on which the gene is situated or the number of copies of a gene present in a given genome, they can utilize this kind of probe.

• Composing multiple smaller probes, each of which binds to a distinct sequence along a chromosome's length, are truly whole chromosome probes. Scientists can give each chromosome a distinct color by labeling them with a variety of fluorescent dyes using several probes. A spectral karyotype is the name given to the final full-color chromosomal map.

• Microarrays have largely taken the place of FISH in many applications. FISH is still helpful for some testing, though. Comparing the gene chromosomal configurations in comparable species is another application for FISH.

What Is GISH?

GISH (genomic in situ hybridization) is a process in which genomic DNAs Are utilized. This method uses unlabeled DNA from the other species being tested as the competition at a much higher concentration, and genomic DNA from one species is used as the labeled probe. At the molecular level, the method is highly helpful for cytologically identifying foreign chromatin in interspecific hybrids. GISH has been utilized in plant molecular cytogenetics to identify parental genomes in translocations and alien segments in naturally occurring allopolyploid species such as Nicotiana tabacum, Triticum aestivum, Millium montianum, and Aegilops triuncialis.

Conclusion

The most reliable method for identifying specific DNA sequences, diagnosing genetic disorders, mapping genes, and identifying novel oncogenes or genetic abnormalities causing different kinds of cancers is fluorescence in situ hybridization or FISH. In fluorescence inverted microscope (FISH) analysis, target sequences of the sample DNA are annealed to fluorescent reporter molecules using DNA or RNA probes. Recently, the technique has been extended to allow simultaneous screening of the entire genome using multicolor whole chromosome probe techniques like spectral karyotyping or multiplex FISH or via an array-based approach utilizing comparative genomic hybridization.