Use of haptenised nucleic acid probes in fluorescent in situ hybridisation

Summary

General

With the advent in this decade of various nucleic acid labelling techniques which allow a non-radioactive visualisation of hybridisation results, the technique of in situ hybridisation, originally developed by Pardue & Gall (1969) and Jones & coworkers (John et al., 1969) have become increasingly important in many biomedical fields. Next to the environmentally important feature of absence of radiation hazards, favourable properties of the non-radioactive techniques such as the high topological resolution attainable, the stability of the labelled nucleic acid probes and the capability to perform multiple hybridisations simultaneously, have contributed significantly to this development.

Haptenisation of probes

Essentially, two types of non-radioactive in situ hybridisation methods can be distinguished: direct methods in which the microscopic reporter molecules, e.g. fluorochromes (Bauman et al., 1980) or enzymes (Renz & Kurz, 1984) are directly coupled to the nucleic acid probe molecules and the indirect techniques in which the nucleic acid probe is labelled with a hapten which, after in situ hybridisation, is detected by immunocytochemical means (Raap et al., 1989).

The advantage of direct techniques is that results can be visualised directly after in situ hybridisation and that potential background staining by nonspecific binding of immunocytochemical reagents does not occur. In contrast with indirect techniques, where the number of reporter molecules can be increased immunocytochemically, they can, however, be limited in their sensitivity. Obviously, when antibodies against the reporter molecule are available (e.g. anti-FITC, Bauman et al., 1981), the favourable properties of both the direct and indirect techniques can be exploited with one nucleic acid labelling procedure.