FISH (Fluorescence in situ Hybridization)

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

*Corresponding author: radharani7737156601@gmail.com

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

• Complementary sequences of target nucleic acids.
• Designed against the sequence of interest.
• Probes are tagged with fluorescent dyes like biotin, fluorescein, Digoxigenin.
• Size ranges from 20-40 bp to 1000 bp.

 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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