Emanuela Boccuni

Fluorescence in situ hybridization (FISH) is a nonradioactive cytogenetic technique which uses fluorescent probes binding only those parts of the chromosome with a high degree of sequence complementarity. It was developed in 1980 and had the goal to identify, and eventually quantify, the presence of given DNA sequences on chromosomes in order to define a spatial-temporal pattern of gene expression. FISH can be also used to detect and localize specific RNA targets, coding and non-coding RNA in cells, tumours and tissue samples.

The possibility to identify the exact positioning of a DNA sequence is the basic concept of in situ approaches. Historically, FISH and other in situ hybridization results played a primary role in mapping genes on human chromosomes. Results from these experiments were collected in databases, and they became very useful for the progress of the Human Genome Project (HGP).

From its development in the 1960s onwards, FISH largely benefitted from improvements of probe-labeling techniques and specific probe design strategies increasing its sensitivity. Its broad application in research and diagnostics are shown by the rise of the number of publications reporting FISH at the beginning of the early 1990s. In recent years, the combination of microfluidic techniques and FISH addresses limitations in probe consumption and hybridization times, making the experimental procedure more sustainable and adaptable to high-throughput developments. Now that the HGP is complete, scientists rarely use in situ hybridization simply to identify the chromosomal location of a human gene: in fact, nowadays there are many applications of this technique, principally focused on clinical diagnoses.

In the figure below, a timeline of fluorescence in situ hybridization developments. The earliest record of in situ hybridization is found by Gall and Pardue in 1969. First fluorescent versions of the technique (FISH) appeared in the 1970s, followed by direct probe labeling twenty years later. ‘Modern’ FISH includes developments in the probe design and production. The combination of microfluidics and FISH first appeared in the early 21st century. Events directly related to the development of FISH are shown in boxes.

In this technique, DNA or RNA probes detect segments of the human genome by DNA-DNA hybridization, DNA sample in a metaphase stage previously treated under conditions that preserve the morphology of condensed human chromosome. Probes are labelled at the 3’ end, usually, with fluorescein or other fluorochromes, that have been replaced the radioactive labels because of their greater safety. Probes can be often derived from an entire sequence of DNA, which has been cut by sequence-specific endonucleases, isolated, purified, and amplified, firstly for use in the Human Genome Project and later for subsequent studies in the same field.

Results are detected by a fluorescence microscopy, used to find out where the fluorescent probe bound to the chromosome: the hybridization reaction generates the fluorescent light.

It is a powerful tool for understanding a variety of chromosomal abnormalities and other genetic mutations. FISH differs from the other techniques for the possibility to perform the procedure both in divided and non-divided cells, making it a very versatile technology.

Moreover, FISH is used for examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities.

FISH APPLICATIONS IN MEDICINE:

in medicine, FISH can have many important applications, in particular it can be useful in:

- Identification of genetic aberrations: in general, aberrations can happen because of DNA breaks that can not be repaired by cellular mechanisms. Normally, from accumulation of these mutations, cancer can arise in human, and, nowadays, abnormalities in about 350 genes have been discovered as implicated in human cancers. Somatic mutations cancer genomes carry two types of somatic mutations: “driver” ones, as a result of selective pressure during tumorgenesis and positively selective, and “passenger” mutations, happening incidentally and  results possible products of genome instability or large number of cell divisions. These last mutations normally lead to transformation of single cell in a detectable cancer.

These aberrations can be detected by FISH and can be therefore used to predict the development of cancer in a specific patient.

It can also be applied in the study of aberrations in prenatal and postanatal patients in order to predict the possible development of a specific disease.

- Detection of aberrations in many different chromosomes at the same time: it is an improvement of FISH application based on the use of fluorescent probes of different colors in order to visualize many different chromosomes together and distinguish them. This is important in the area of Gene Mapping

- Another improvement of application in Gene Mapping is the ability to identify several different regions on the same chromosome by using, also in this case, different probes together. The process is called MULTIPLEX FISH and allow painting of the entire chromosome complement in a single hybridization through labelling each chromosome with a different combination of fluorophores.

- Comparative genomic hybridization to analyze chromosomal imbalance in tumors and to examinate possible correlations between findings and tumor phenotypes

- Study of chromosomal aberrations also in non dividing cells, leading to chromosomal mapping of some deleted or amplified region

- Visualization and measurement of telomere length

- Quantification of gene copy number and the amount of protein

 in general we can conclude saying that FISH in medicine finds its best applications in the correlation between DNA sequences (mutations and modifications inside it) and phenotype, usually cancer or disease. by using this process it can also be possible to predict the onset of a disease in a patient in different stages of his life.

SOURCES: 

“Application of Fluorescence In Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science”

10.7759/cureus.1325

“Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance”

 https://doi.org/10.1093/biohorizons/hzq009