Fluorescence in situ hybridization

FISH (Fluorescence In Situ Hybridization) is a molecular genetic technique applied in cytogenetic examinations to detect small chromosomal rearrangements.

FISH - Technique: Basic steps

 * Preparation of target DNA: DNA of metaphase or interphase cells are denatured into single-stranded DNA


 * A DNA probe, corresponding to a specific chromosomal DNA sequence is labeled with a specific fluorophore


 * Hybridization: Target DNA and the labeled DNA sequence are hybridized in situ to fixed metaphase or prometaphase chromosome spreads on a glass slide


 * Each probe has the possibility of hybridizing specifically to two sister chromatids. The probe, marking a specific sequence of the chromosome is then visualized

Labeling
It is possible to either label the probe directly or indirectly. Direct labeling involves a labeled and modified nucleotide (often 2' deoxyuridine 5' triphosphate), which is directly incorporated into the probe. In indirect labeling, the DNA is labeled with a fluorophore to make the signal visible (not being incorporated into DNA). Indirect labeling can involve e.g. a hapten- or biotin- labeled probe which is then marked by fluorophore labeled antibodies or avidin.

Types of probes

 * 1) Centromeric (satellite probes)
 * 2) Locus specific probes
 * 3) Whole chromosome painting probes (e.g. used in mFISH)



Which chromosomal aberrations can be identified?

 * translocations (balanced and unbalanced)
 * copy numer changes
 * additions
 * deletions
 * insertions
 * inversions
 * identifies chromosomal origins
 * can identify specific p/q arms/bands

FISH vs. traditional Karyotyping
Traditional karyotyping allows scientists to view the full set of human chromosomes. Usually G-banding (Giemsa- stain) is used to display the bands of the chromosomes in a black and white pattern. Interpretation might be quite difficult, because the resolution is not always sufficient and usually an expert is needed, who might need a long time interpreting the bands.

FISH uses fluorescent dyes, which then can be painted with a specific computer program, so that even non-experts can easily see instances where e.g. a chromosome has parts of an other chromosome attached to it.