Fluorescence in situ hybridization (FISH) is a method used to detect specific DNA sequences in chromosomes. The technique uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence similarity to the probe. This makes it possible to visualize individual chromosomes and specific regions within chromosomes.
FISH can be used to detect chromosomal abnormalities, such as aneuploidy (too many or too few chromosomes) translocations, inversions, and deletions. It can also be used to identify genes that are associated with certain diseases. In addition, FISH can be used to map the locations of genes on chromosomes.
Steps in FISH Analysis
- Probe Preparation: DNase is added to introduce random nicks (cuts) in the DNA. Special nucleotides with attached fluorophore are added followed by the addition of ligase to seal the nicks
- Denaturation: The probe and target DNA are heated to 95oC to separate the stands.
- Cooling: The DNA is cooled to allow the probe to bind to the target DNA sequence
- Washing: Probe that did not bind to the DNA are washed away
- Analysis: The results are observed under a fluorescent microscope
If the probe is designed that it can only bind if it is 100% complementary to a gene, you will see a stained location on the gene under the microscope. However, if there is a mutation, no hybridization is possible. Short probes are designed to detect diseases that are caused by a gene mutation. Long probes may be developed to stain the entire chromosome and stain them in different colors. Trisomy 21, a condition that causes down syndrome can be easy detected using this method.