FISH

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used to detect the presence of a specific DNA sequence on a chromosome.

The technique is based on the use of a probe: a short DNA sequence (10-25 nucleotides), specifically designed for the target sequence. To allow the detection of a wanted genic locus the probe is fluorescent-labelled (direct labelling) or can be rendered fluorescent in a subsequent phase (indirect labelling).

The first protocol step is the attachment of either interphasic or metaphasic chromosomes on a solid surface (glass); then the sample and probe solution is denatured and incubation starts. During the incubation period (hours, depending on the protocol), target DNA and probe interact and hybridize. To avoid background, unbound/partially bound probes are washed away, by repeated washes, after incubation.

The detection step can be different: in case of direct detection the probe is already fluo-marked and the sample can be directly observed at fluorescence microscope. On the contrary, in the indirect labelling it is necessary to add an enzymatic or immunological detection system, which will render the probe fluorescent. This method needs more time but allows to amplify the signal and get a better detection.

FISH is a versatile technique: can be useful to detect a gene locus on a chromosome, chromosomal abnormalities and rearrangements. Nowadays several connected techniques have been developed starting form the “classical” FISH, which is focused on chromosomal loci.

 (Giada Cipollina)

Immuno-FISH

RNA-FISH

others....