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Nucleic Acids Research, 1986, Vol. 14, No. 16 6471-6488
© 1986

Articles

A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands

A.H.N. Hopman*, J. Wiegant, G.I. Tesser+ and P.Van Duijn

Department of Histochemistry and Cytochemistry, University of Leiden Wassenaarseweg 72, 2333 AL Leiden +Department of Organic Chemistry, Catholic University of Nijmegen Toernooiveld,6525 ED Nijmegen, The Netherlands

*To whom correspondence should be addressed

Received June 3, 1986. Revised July 29, 1986. Accepted July 29, 1986.

Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to rcercurated nucleic acids, were immunocher.ically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dotblot hybridization reactions on nitrocellulose.


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