Open AccessA non-radioactivein situhybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands
Author(s) -
Anton H. N. Hopman,
J. Wiegant,
G. I. Tesser,
P. van Duijn
Publication year - 1986
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/14.16.6471
Subject(s) - hapten , nucleic acid , nitrocellulose , biology , in situ , nucleic acid thermodynamics , dna , pyrimidine , ligand (biochemistry) , biochemistry , in situ hybridization , hybridization probe , microbiology and biotechnology , oligonucleotide , amino acid , chemistry , antibody , organic chemistry , receptor , base sequence , membrane , genetics , gene , gene expression
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 mercurated nucleic acids, were immunochemically 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 dot-blot hybridization reactions on nitrocellulose.
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