Fluorescence in situ hybridization uses fluorescently labeled DNA probes to detect and localize specific DNA sequences on metaphase chromosomes and in interphase nuclei, enabling direct visualization of genomic organization and abnormalities.
Principle
FISH follows the same hybridization principles as colorimetric in situ hybridization but uses fluorophore-conjugated probes for direct detection without enzymatic amplification. Fluorescent signals are visualized by epifluorescence or confocal microscopy. Multiple probes with distinct fluorophores can be applied simultaneously, enabling multicolor analysis of several genomic loci in a single specimen.
Probe Types
Locus-specific probes target unique genomic regions, typically 50–500 kb cloned into BACs or fosmids. They are labeled by nick translation with fluorescent nucleotides such as SpectrumOrange, SpectrumGreen, or Cy5. Centromeric probes target alpha-satellite repeat sequences and identify specific chromosomes. Telomeric probes label chromosome ends. Whole-chromosome painting probes are complex mixtures of sequences unique to a single chromosome, generated by flow sorting or microdissection. Oligonucleotide-based probe sets synthesized on microarrays now offer customizable, high-resolution coverage.
Labeling Methods
Direct labeling incorporates fluorophore-conjugated nucleotides during probe synthesis. Detection is immediate after hybridization. Indirect labeling incorporates haptens such as digoxigenin or biotin, which are detected after hybridization with fluorophore-conjugated antibodies or streptavidin. Indirect methods provide signal amplification through antibody layers and are useful for small targets. Tyramide signal amplification further increases sensitivity.
Sample Preparation
Metaphase chromosome spreads are prepared from colcemid-arrested cell cultures, hypotonically swollen, and fixed in methanol:acetic acid (3:1). Interphase nuclei are obtained from uncultured cells or fixed tissues. Specimens are aged, treated with RNase and pepsin to reduce background, and dehydrated through an ethanol series. Denaturation of target DNA in 70% formamide at 72 °C is required for double-stranded probes. Formamide-free protocols using heat denaturation in hybridization buffer are alternatives.
Hybridization and Washing
Probe and target are co-denatured at 75 °C for 5 minutes, then hybridized at 37 °C for 4–16 hours in a humidified chamber. Post-hybridization washes in SSC and Tween-20 remove unbound probe. Stringency is controlled by formamide concentration, wash temperature, and salt concentration. After washes, slides are counterstained with DAPI, which produces a Q-banding pattern on metaphase chromosomes for karyotype identification.
Multicolor FISH
Multiplex FISH (M-FISH) uses combinations of five fluorophores in a combinatorial labeling scheme to generate 24 chromosome-specific probe sets, each with a unique spectral signature. Spectral karyotyping uses a similar approach with imaging spectrometry. These techniques enable comprehensive karyotype analysis in a single experiment, detecting complex rearrangements, marker chromosomes, and cryptic translocations.
Applications
FISH is a clinical mainstay for detecting chromosomal aneuploidies in prenatal diagnosis using uncultured amniocytes and chorionic villus samples. HER2 gene amplification status in breast cancer is routinely assessed by FISH. BCR-ABL fusion detection in chronic myeloid leukemia uses dual-color, dual-fusion probes on interphase nuclei. The Philadelphia chromosome t(9;22) appears as a fusion signal. FISH on formalin-fixed, paraffin-embedded tissue sections maps genomic changes in solid tumors. Chromosome-specific paints are essential in radiation biology for quantifying DNA damage and repair through micronucleus and translocation assays.
