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.