A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands

Abstract

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achieved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand. In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands. The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.