Labelling of probes

The detection of homologous sequences after hybridization with the probe is like finding a needle in the haystack. Therefore, for the success of DNA probe assay, it is necessary to develop simple, safe and sensitive techniques for their use. As probes transmit no signal of their own, they have to be (i) labelled either with radioactive isotopes (e.g. ³²P dCTP) or with non-radioactive signal molecules (e.g. biotin or digoxigenin) without impairing the hybridization ability of these probes. These signal molecules may include fluorescent antibodies and enzymes that produce colour changes in dyes and chemiluminescent catalyst, (fig 39.16).

After hybridization with radioactively labelled probe, hybrids are detected by autoradiography (Fig. 39. 3). ³²P has the advantage over other radioisotopes, since it has high specific activity. However, in general, radioisotopes have some disadvantages. They are difficult to handle and expensive to dispose off. Detection by autoradiography, while sensitive, may take a long time if there are few counts in the hybrid. Furthermore, radioisotopes have a short half life (e.g. ³²P has a half-life of 14.3 days) and therefore experiments should be completed preferably within one half-life.

Recent advances in nucleic acid technology now offer alternatives to radioactively labelled probes. One procedure that is becoming increasingly popular is biotin labelling of nucleic acids. After hybridization and washing, detection of hybrids is done by a series of cytochemical reactions which finally gives a blue colour whose intensity is proportional to the amount of biotin in the hybrid. There are several advantages of using biotinylated probes. For example, these assays employ non-toxic materials, whose half-life is longer. These probes can be prepared in advance in bulk and stored at - 20°C for repeated uses.

Digoxigenin is another chemical derived from plants and used for non- radioactive labelling of probes. An antibody associated with an enzyme (anti- digoxigenin-alkaline phosphatase conjugate) is used for the detection of the presence of digoxigenin. The probe may be labelled with digoxigenin-11-dUTP supplied with a digoxigenin kit (these kits are available from any commercial firm, e.g. Boehringer Mannheim). The labelled and denatured probe may be used for hybridization with denatured DNA on Southern blots or for in-situ hybridization. After hybridization the membrane or the slide may be washed and the membrane or the slide is transferred into detection buffer containing 20 μg/ml of anti-digoxigenin fluorescein and 5% (w/v) BSA (bovine serum albumin). The system is incubated for 1h at 37°C, and then the membrane/slide is washed in detection buffer three times (8 min each at 37°C). Alkaline phosphatase activity can be detected using 017 mg/ml BCIP (5-bromo 4-chloro 3-indolyl phosphate) and 0-33 mg/ml NBL (nitroblue tetrazolium) as dye substrate