VPB 321: Animal Biotechnology (2+1)

ISOTOPIC AND NON- ISOTOPIC LABELING SYSTEMS

Isotopic labeling systems

• Traditionally, nucleic acids have been labeled with radioisotopes such as ³²P and ³⁵S which are detected by autoradiography. These radiolabeled probes (hot probes) are very sensitive but their handling is subject to strict safety precautions and the signal decays relatively quickly.

Non-isotopic probes (cold probes)

• Non-isotopic probes (cold probes) generate colorimetric or chemiluminescent signals. A widely used label is digoxigenin, a plant steroid isolated from digitalis.
• This can be conjugated to nucleotides and incorporated into DNA, RNA or oligonucleotide probes and then detected using an antibody.
• Another system uses biotin, a vitamin, and the bacterial protein streptavidin which binds to biotin. Biotin conjugated nucleotides are incorporated as a label and detected using streptavidin.
• The presence of a target nucleotide sequence in a DNA sample can be determined with a DNA probe. This procedure is called DNA hybridization and depends on the formation of stale base pairs between the probe and the target sequence.
• For a DNA hybridization assay, the target DNA is denatured and the single strands are irreversibly bound to a matrix (e.g. nitrocellulose or nylon), this process is often carried out at a high temperature.
• Then, the DNA probe which is labeled with either a radioisotope (hot probe) or another tagging system (cold probe) is incubated with the bound DNA sample.
• If the sequence of nucleotides in the DNA probe is complementary to a nucleotide sequence in the sample, then base pairing (i.e. hybridization) occurs.
• The hybridization can be detected by autoradiography (hot probe) or other visualization procedures (cold probe), depending on the nature of the probe label.
• If the nucleotide sequence of the probe does not base pair (bind) with a DNA sequence in the sample, then no hybridization occurs and the assay gives a negative results.