Labeling DNA Probes

Imagine an experiment where the goal is establishing the presence or absence of a specific gene in a bacterial genome. How might such a small nucleic acid sequence be detected within the complex genomic DNA?

DNA probes can be used to achieve this - these are single-stranded DNA molecules that are complementary to the target nucleic acid sequence and labeled with a detection molecule.

The most commonly used labels for DNA probes include radioisotopes like Phosphorus 32, fluorophores like fluorescein, or small molecule binding partners such as biotin and digoxigenin.

These labels can be incorporated chemically after the synthesis of DNA probes or during their synthesis via one of the two methods.

In the end-labeling method, the hydroxyl or phosphate of the nucleotide present at the 5’ or 3’ termini of the probe DNA sequence is covalently bound to the desired chemical group or label.

For internal labeling, the nick translation method uses the DNase enzyme to make a small nick in one of the DNA strands followed by DNA polymerase I - which adds labeled and unlabeled nucleotides directly into the DNA strand. This results in a probe where one or more of the nucleotides is labeled – such a