NON-RADIOACTIVE IN SITU HYBRIDIZATION PROCEDURES USING MERCURY- AND ACETYLAMINOFLUORENE LABELLED PROBES

抄録

Two methods for nucleic acid hybridization are discussed in which, instead of radioactive markers, labels like fluorochromes or cytochemically detectable enzymes are applied. In one method mercury is covalently bound to the probe in order to allow, after the hybridization, coupling of a ligand which in addition to the mercury-binding thio-group, contains a fluorochrome or a hapten. In another procedure the probe is haptenized before the hybridization by treatment with N-acetoxy-2-acetylaminofluorene, resulting in the covalent attachment of AAF-groups.
In both methods, the haptens in the hybrids are visualized by immunocytochemical procedures.
Our efforts so far, resulted in methods which in addition to speed and a high topological resolution, reached a sensitivity that allows the detection of unique mammalian sequences in both in situ and in Southern blot hybridization experiments. With these procedures the detection of separate target DNAs simultaneously within one microscopic preparation is possible, as is in situ hybridization on interphase nuclei in suspension. Several aspects of these procedures as well as results obtained so far are discussed.