Immunoenzymatic techniques applied to the specific detection of nucleic acids: A review

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorophores, lumiphores can be attached to the nucleic acid probe either by covalent bonds (direct labelling) or by biospecific recognition after hybridization (indirect labelling). The principle of the latter method is based on the use of a hapten-labelled nucleic acid probe which is generally detected by an immunoenzymatic assay. Indirect labelling has several advantages: this procedure uses multienzyme complexes to increase the number of enzyme molecules associated with hybridization and hence provides an increase in detectability; moreover, haptens (biotin, dinitrophenol, acetylaminofluorene analogues, digoxigenin, brominated or sulphonylated pyrimidines) used to label nucleic acid probes are not sensitive to elevated temperatures (42–80°C), extended incubation times (several hours), ddetergent and organic solvents currently required in hybridization techniques. The application of the immunoenzymatic and related techniques to nucleic acid probing is reviewed, focussing on the strategies of non-radioactive hybridization, hapten-labelling of nucleic acids and methods for the immunodetection of the hybrids.