FISH (Fluorescence in situ Hybridization)

February 2, 2024

FISH (Fluorescence in situ Hybridization)

Radha Rani Sawami^1*, Vaishali Pareek¹, Nitesh Jat¹, Anju Kumari¹, Yashmita Shekhawat¹

¹Department of Animal Genetics and Breeding, College of Veterinary and Animal Science, Bikaner, RAJUVAS

INTRODUCTION

FISH is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarities.
Is used to detect and localize the presence or absence of specific DNA sequences on chromosomes affixed to a microscope slide .
Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes.

PRINCIPLE:

The basic principle involved is hybridization of nuclear DNA either interphase cells or of metaphase chromosomes affixed to a microscopic slide, with a nucleic acid probe.
A DNA probe is tagged with a fluorescent marker. The probe and target DNA are denatured, and the probe is allowed to hybridize with the target. The fluorescent tag is then detected with a fluorescent microscope.

PROBES

TYPES OF PROBES:

Whole chromosome painting probes: It is a mixture of probes that binds to the entire chromosome length and thus different chromosomes are colored or labeled with different colored probes.
Centromere probes: Generated from repetitive sequences found in centromeres. Centromere regions are stained brighter.
Telomere probes: Specific for telomeres. Probe is based on the TTAGGG repeat present on all human telomeres.
Locus specific probes: bind to a particular region of chromosome. It is useful for detecting structural rearrangements such as specific chromosomal translocations, inversions or deletions in both metaphase and interphase.

BASIC PROCEDURE

The DNA probe is labelled indirectly with a hapten (left panel) or directly labelled via the incorporation of fluorophore (right panel).
The labeled probe and the target DNA are denatured. Combining the denatured probe and target allows the annealing of complementary DNA sequences.
If the probe has been labelled indirectly, an extra step is required for visualization of non- fluorescent hapten that uses an enzymatic or immunological detection system. Finally, the signals are evaluated by fluoroscence microscopy.

ADVANTAGES OF FISH

Cell culture process is not needed for performing FISH.
In the conventional karyotyping method, scientists must have to culture chromosomes and arrest them on metaphase.
The fluorescence in situ hybridization method is rapid and the chance of contamination is negligible.
The karyotyping method is entirely based on the chromosome banding thus it is restricted, using multiple probes in FISH multiple hybridization sites have been analyzed using different fluorophores.
Not only metaphase but also the interphase chromosomes can also be used in FISH in order to achieve higher resolution.
Another advantage of FISH is it allows the analysis of the non-dividing cells such as solid tumor cells. Efficiency of hybridization and deletion is high.
Sensitivity and specificity is high.

LIMITATIONS OF FISH

Probe design requires knowledge of specific chromosomal abnormalities to be studied.
Cutoff signals may be different among laboratories.
Processing errors, imperfect hybridization, non-specific binding, photo bleaching, inter-observer variability, and false positive and negative results are possible.

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