-Fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH)

Using Fluorescent-labelled DNA probes which are designed that are complementary to the DNA sequences being assessed, to detect and localize the presence or absence of specific DNA sequence on Chromosome.

Steps:

ü Chromosomes are immobilized and denatured on a microscope slide and exposed to a solution containing a fluorescently labelled probe specific to a specific chromosomal region.

ü After hybridization (the formation of a double strand of DNA from complementary single strands), the slide is washed and examined microscopically.

ü Where the probe has hybridized, fluorescent spots are seen over the relevant chromosome.

For example, if a child were suspected of having 22q11 deletion syndrome, FISH using a 22q11-specific probe would show only one pair of fluorescent spots, rather than two.

Types of FISH probes:

ü  Centromeric (aneuploidy)

ü  Telomeric (subtelomeric rearrangements)

ü  Sequence specific (microdeletions)

ü  Whole chromosome paint (complex rearrangements)

ü  Reverse painting (to identify origin of unidentified chromosomal material)

Useful for microdeletion syndromes.

FISH can be performed much more rapidly than formal karyotyping.

As FISH testing only detects abnormalities in the region targeted by the chosen probe, it requires clinical recognition of the likely causative mechanism. For this reason, the use of MLPA, Qf-PCR and CGH microarray has largely superseded this process, except in testing other members of a family for a known chromosomal anomaly.

It is now most commonly used for rapid testing for aneuploidy (chromosome number not a multiple of the haploid number, i.e. of 23 chromosomes in humans) and for the follow-up of array-detected abnormalities.