AAT Bioquest

What are the general steps of fluorescence in situ hybridization (FISH) experiments?

Posted July 22, 2020


The FISH experiment generally contains 5 steps:

  • Preparation of probes: Short sequences of single-stranded DNA that are complementary to a portion of the target sequence are prepared (usually by cloning).
  • Probe labeling: The probes are labeled prior hybridization, which can be achieved by various means, such as nick translation, random primed labeling and PCR. Probes can either be directly labeled with a fluorophore, or indirectly labeled with a hapten that can later be recognized with an enzymatic or immunological detection system.
  • Denaturation: Both the target and probe sequences are denatured with heat or chemicals.
  • Hybridization: The target and probe sequences are combined and annealed, allowing the hybridization of complementary sequences.
  • Visualization: Target sequences are visualized. If the probe is indirectly labeled with the nonfluorescent hapten, an extra step is required to visualize the hapten using an enzymatic or immunological detection system.
Additional resources

Helixyte™ Green *10,000X Aqueous PCR Solution*

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Levsky, J. M., & Singer, R. H. (2003). Fluorescence in situ hybridization: past, present and future. Journal of cell science, 116(14), 2833-2838.