15.4: Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via end-labeling, nick-translation, or random primer synthesis methods. However, it is essential to bear in mind the intended application of the probe or the source of target nucleic acid and the choice of label when designing a labeling strategy.
The probe hybridized to the target molecule can be detected by one of a whole range of methods, depending on the type of label used. For example, fluorophores can be directly visualized through their fluorescence. Alternatively, small molecule binding partners like digoxigenin can be detected using high-affinity Anti-digoxigenin antibodies coupled to a reporter tag, such as a fluorophore or an enzyme.
Application of DNA Probes
DNA probes are widely used in molecular biology methods, including screening gene libraries, detecting nucleotide sequences with blotting methods, or gene technologies such as nucleic acid and tissue microarrays. They can also be used for purification of interacting molecules like DNA binding proteins. In environmental or health studies, DNA probes can be used for the detection of specific genes and microorganisms in environmental or pathological samples using in-situ hybridization.