Fluorescence in Situ Hybridization: Cell-based Genetic Diagnostic and Research Applications

Introduction to Fluorescence In-Situ Hybridization (FISH)

Fluorescence in-situ hybridization (FISH) is a molecular diagnostic technique that allows visualization of specific chromosome nucleic acid sequences within a cellular preparation. It involves the precise annealing of a fluorescence-labeled DNA probe to complementary targeted sequences. Thus, the genes or sequences of interest can be observed visually using a fluorescence microscope. The most basic components of FISH are the targeted DNA sequence and the DNA probe. Before hybridization can occur, the DNA probe must be labeled using various methods, such as random primed labeling, nick translation, or polymerase chain reaction. Two different strategies can be used for labeling: the direct method and the indirect method.

Labeling Techniques in FISH

In indirect labeling, the DNA probe is labeled with an altered nucleotide that contains a hapten, while in direct labeling, nucleotides that have been directly altered to contain a fluorophore are used. These fluorescently labeled probes, i.e., FISH probes, are denatured first, and subsequently, the combination of the denatured probe and target sequence allows the complementary DNA sequences to anneal. For the probes that have been labeled indirectly, an additional step is required to visualize the non-fluorescent hapten, which often uses an enzymatic or immunological detection system. Although FISH is faster with the use of directly labeled probes, indirect labeling offers the benefit of signal intensification by using multiple layers of antibodies. Consequently, brighter signal production may occur compared to background levels.

Applications of FISH in Molecular Diagnostics

Due to its ability to detect chromosomal aberrations such as gene rearrangement, gene deletion, and gene amplification, as well as its high specificity, FISH procedures are widely used in molecular diagnostics to detect and identify:

• Centromeres of a specific chromosome.
• Specific oncogenes (locus-specific probe, useful for ROS1, ALK), amplifications (HER-2), deletions (CLL), etc.
• Specific tumor-suppressor genes (locus-specific probe, loss is relevant to tumor progression).
• Whole chromosomes (useful for detection of complex chromosomal rearrangements).

FISH procedures are generally carried out on FFPE (Formalin-fixed paraffin-embedded) tissue preparations or cellular preparations. Advantages of FISH analysis compared to conventional chromosomal analysis include the following:

• A large number of cells can be examined, which is helpful in the detection of residual disease.
• Metaphasic cells are not essential, so aberrations can also be detected in non-dividing cells, which is beneficial in chronic lymphocytic leukemia.
• FISH can be executed in a relatively short amount of time.
• Aberrations that are too subtle to be detected by conventional cytogenetic analysis can also be identified.

FISH Procedure and Observations

Most FISH procedures are carried out in the dark to avoid photo-bleaching, as probes are highly sensitive to light. The FISH slides are observed under a fluorescence microscope fitted with excitation and dichroic filters specific to the probe. The FISH procedure, as performed during an internship, is as follows:

1. Incubate the FFPE (formalin-fixed paraffin-embedded tissue) at 70°C for 3 hours, followed by dewaxing in xylene for 10 minutes (twice).
2. Dehydrate the slides in 70%, 85%, and 100% ethanol for 3 minutes each, followed by air drying.
3. Immerse the slides in 0.2M HCL for 20 minutes.
4. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each.
5. Immerse the slides in 1M NaSCN for 30 minutes at 80°C.
6. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each.
7. Immerse the slides in 3.5% pepsin solution at 37°C for 10 minutes.
8. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each.
9. Immerse the slides in 10% neutral buffered formalin for 10 minutes.
10. Immerse the slides in molecular water followed by 2X SSC (sodium saline citrate) for 2 minutes each.
11. Dehydrate the slides in 70%, 85%, and 100% ethanol for 3 minutes each, followed by air drying.
12. Add 5µl of the appropriate probe onto the slides.
13. Incubate the slides in Thermobrite© pre-set for hybridization.
14. Immerse the slides in wash solutions for 3 minutes each.
15. Air dry in the dark and add 5µl of DAPI + Antifade.
16. Observe the slides under a fluorescence microscope.

Conclusion

Fluorescence in-situ hybridization is a powerful tool in molecular diagnostics due to its ability to provide detailed insights into chromosomal and genetic abnormalities. Its applications range from cancer diagnostics to genetic research, offering precise and rapid results. As molecular techniques continue to advance, FISH remains a cornerstone method for understanding complex genomic landscapes (Langer-Safer et al., 1982; Pinkel et al., 1986).

References

Langer-Safer, P. R., Levine, M., & Ward, D. C. (1982). Immunological method for mapping genes on Drosophila polytene chromosomes. Proceedings of the National Academy of Sciences of the United States of America, 79(14), 4381-4385.

Pinkel, D., Straume, T., & Gray, J. W. (1986). Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proceedings of the National Academy of Sciences of the United States of America, 83(9), 2934-2938.