How can the hybridization probes be labeled?

Before hybridization, double-stranded DNA probes must undergo denaturation to separate the two DNA strands, while single-stranded oligonucleotide and RNA probes do not require denaturation. Probes can be labeled with radioactive isotopes through two main methods: nick translation and the random priming technique. In nick translation, the process begins with the DNA probe being fragmented by a DNAse enzyme, creating nicks or single-stranded regions in the DNA. Then, DNA polymerase I, along with radioactive nucleotides (typically labeled with 32P), fills in the gaps by extending the DNA strands from the nick sites. As the DNA polymerase incorporates the radioactive nucleotides into the probe, it becomes labeled. In the random priming technique, primers are annealed to the single-stranded DNA template. These primers are usually 6-12 nucleotides in length and contain a radioactive or fluorescent label at their 5' end. DNA polymerase, along with radioactive nucleotides, extend the primers along the DNA template in a random manner; this generates labeled DNA fragments of varying length. Labeling typically occurs at the 5' end using 32P, achieved with the enzyme bacteriophage T4 polynucleotide kinase and gamma-labeled ATP.