FISH - Fluorescent In-situ Hybridization

Fluorescence In situ Hybridization or FISH is a technique used to detect the location of specific genes or parts of genes on chromosomes within fixed cells.

In this technique, a fluorescently labeled single-stranded DNA is used as a probe to bind parts of chromosomes that contain a complementary DNA sequence, referred to as the target.

First, cells are arrested in interphase or metaphase. The arrested cells are suspended in buffer solution and spread on a clean glass slide.

Next, the target DNA and the labeled probes are denatured with heat or chemicals. This step separates the double stranded DNA leaving both the probe and the target DNA as single-stranded sequences that are capable of base pairing.

Then, the probe and target DNA are mixed together and incubated overnight. This allows the probe to hybridize to its complementary sequence on the chromosomes.

Once hybridization is complete, the excess probe is washed off.

If the probe was labeled with a fluorophore, the locations of the hybridized DNA can be directly visualized with a fluorescent microscope.

If not, the hybridized DNA needs to be fluorescently labeled before visualization.  To do so, the probe sequence must contain a modified nucleotide bonded to a hapten– a small molecule to which the fluorophore—such as a fluorescent antibody, can attach.

Such direct visualization of genes is very useful for cell-based diagnostic assays, where genetic abnormalities can be detected from any loss..., gain..., or rearrangement of chromosomes during cell division.