Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).
A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.
Kessler
C
C
Boehringer Mannheim GmbH, Biochemisches Forschungszentrum Penzberg, Tutzing.
Höltke
H J
HJ
Seibl
R
R
Burg
J
J
Mühlegger
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000378:metabolism; D015139:Blotting, Southern; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D015342:DNA Probes; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005057:Eukaryotic Cells / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D009696:Nucleic Acids / Q000032:analysis*; D010761:Phosphorus Radioisotopes
D000906:Antibodies; D015342:DNA Probes; D009696:Nucleic Acids; D010761:Phosphorus Radioisotopes; D001710:Biotin; D004247:DNA; D004076:Digoxigenin