Hello! I am curious about the minimum hybridization time for the custom locus-specific probes for FISH. I know that it should take at least 12 hours at 37 degrees Celsius, but is there an even lower threshold when you can get results?
Hello Philipp Spartakovich Sviridov
The hybridization time in FISH may range from 60 minutes to 24 hours. Hybridization specificity is driven by the degree of complementarity between the probe and target sequences, as well as by the probe length. These are the characteristics that will directly influence probe concentration, temperature and time of hybridization. Moreover, hybridization buffer can also influence in situ hybridization efficiency. There are fast-working hybridization buffers that are commercially available which can shorten the incubation time significantly from an overnight incubation to a few hours.
The standard formamide FISH hybridization buffer lowers the melting temperature of nucleic acids by attacking hydrogen bonds and interfering with DNA base pairing. This interference slows down base pairing between the FISH probe and the DNA target in the specimen. However, fast-working hybridization buffer (like the one provided in the link below) destabilizes the DNA helix by diminishing hydrophobic base stacking. Minimized base pairing interference enables fast binding between the FISH probe and specimen DNA. It allows the hybridization phase of a FISH assay to be completed in 1-2 hours and the entire assay can be completed in half a day. Moreover, it can be used with any nucleic acid probe.
Please refer to the link below.
https://www.agilent.com/cs/library/brochures/29145_iqfish_fast_hybrid_brochure.pdf
Best.
Malcolm Nobre.
Thank you for your answer and information about Agilent's product. I have never heard of it and it would be very interesting to try.
Best regards,
Philipp S.
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