Fluorescent In Situ Hybridization (FISH) EXPLAINED

Fluorescent in situ hybridization, or FISH, can be used in order to visualize specific locations on a chromosome and even detect irregularities in the DNA of the chromosome. The FISH-Assay is carried out in 4 main steps: 1. First, the cell has to be fixated using formaldehyde which causes extensive cross-linking between the proteins of the cell and the assay. 2. Second, the probe is designed by first taking a DNA strand which is complementary to the chromosomal region of interest. Then DNase which is an endonuclease, induces random “nicks” or cuts in the probe. Now DNA polymerase 1, can attach itself to the OH-end of these nicks to begin translation and incorporation of these fluorescently labeled nucleotides. Now, we have a complementary labeled probe. This probe can now be amplified using PCR. 3. Third, both probe and the chromosomal target DNA are denatured by heating up the cell to around 95 degrees Celsius. 4. Finally, hybridization can take place once the cell has cooled down, allowing the probe to specifically bind to the target DNA sequence. Once bonding has occurred, all probes not bond are washed away. It is VITAL to note that hybridization can NOT occur UNLESS the probe is IDENTICAL to the target DNA sequence. In other words, any mutations can be detected because the chromosome will NOT be fluorescently marked. Now, as we can see in this case, the chromosome “glows” in a specific section, meaning that the probe has successfully bond to the target DNA. Here, on the other hand, the chromosome does not glow, meaning that a mutation has occurred.