Fluorescence in situ hybridisation (FISH)
FISH uses a fluorescent probe, a short piece of DNA or RNA tagged with a dye, to show where a chosen sequence sits in cells, even non-dividing ones.
Remember
A fluorescent probe finds and locates its chosen sequence in place, so FISH works in non-dividing cells and does not need cultured, dividing cells as karyotyping does.
Memory aid
The probe goes fishing for its matching sequence, and the fluorescent glow shows where it was caught.
Picture the probe as fishing for its one matching sequence, with the glow marking where it was caught.
Fluorescence in situ hybridisation, or FISH, detects and locates a chosen DNA or RNA sequence (the order of bases) in cells on a slide. In situ means in its original place, so FISH shows where a gene or region lies.
The tool is a probe: a short piece of DNA, or RNA, tagged with a fluorescent dye. The probe is built to be complementary to the target, which means its bases pair up with the target's bases. When it is added to cells fixed on a slide, it hybridises, which means it pairs up by complementary bases, and it does so only with its matching sequence. Under a fluorescence microscope, the glowing spot shows whether the sequence is present and where it sits.
The main advantage over karyotyping (arranging a cell's chromosomes by size and banding to look for changes in number or structure) is that FISH can be applied to interphase cells, which are cells that are not dividing. Karyotyping needs dividing cells that have been cultured and stopped at metaphase (the stage of cell division when chromosomes are most condensed).
This is very useful in cancer. A tumour sample with few dividing cells can still be studied. An amplified gene (one copied many times over) lights up as extra bright spots, as with HER2 in breast cancer. A probe can also show that a gene has moved to another chromosome, as in the BCR-ABL rearrangement of chronic myeloid leukaemia (a blood cancer in which two genes are joined together abnormally).
FISH is targeted. It looks only at the sequence the probe was designed for, so it is not a screen of the whole genome.
Key facts
- Probe
- a fluorescent DNA probe pairs only with its matching sequence in cells on a slide.
- Cells needed
- FISH works in interphase nuclei, so actively dividing cells are not required.
- Comparison
- karyotyping needs cultured dividing cells stopped at metaphase.
- What it shows
- the position and number of copies of a chosen gene, not the amount of protein.
- Limit
- FISH looks only at the sequences its probes match, so one normal probe result does not exclude every other abnormality.
Exam traps
- Working out the exact nucleotide sequence of a gene needs sequencing, because FISH only shows whether a probe has found its matching sequence and where, not the order of bases.
- Copying DNA exponentially so that it can be sequenced describes PCR, not FISH, because FISH does not make more DNA and only shows where a labelled probe has bound.
- G-banding is the staining pattern used in standard karyotyping, in which chromosomes are viewed during metaphase, so it is a feature of the method FISH is compared with and not an advantage of FISH.
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