AAT Bioquest

What methods are commonly used to label the probes in fluorescence in situ hybridization (FISH)?

Posted July 22, 2020


Three methods are commonly used to label the nucleic acid (DNA and RNA) probes with desired fluorophores, including PCR labeling, nick translation, random oligo primed synthesis, and end labeling.

  • PCR labeling: It is a straightforward and now very popular method to incorporate labeled nucleotides into a probe, which can be accomplished simply by using a labeled dNTP during PCR.
  • Nick translation: This method involves 2 enzymes: DNase I and DNA polymerase I. Nicks (i.e. single strand breaks) are first introduced by DNase I to the DNA backbone. Then DNA polymerase I removes nucleotides by 5’-3’ exonuclease activity and replaces them with modified dNTPs, thus labeling the probe.
  • Random oligo primed synthesis: DNA is first denatured in to single strands, followed by annealing with random hexamer oligonucleotides for hybridization. DNA polymerase is then used to extend these random primers, incorporating labeled nucleotides.
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.