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.