NDLI: 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)

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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)

Content Provider	Scilit
Author	Kessler, Christoph Höltke, Hans-Joachim Seibl, Rudolf Burg, Josef Mühlegger, Klaus
Copyright Year	1990
Abstract	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.
Related Links	https://www.degruyter.com/downloadpdf/journals/bchm/371/2/article-p917.pdf
Ending Page	928
Page Count	12
Starting Page	917
ISSN	01773593
DOI	10.1515/bchm3.1990.371.2.917
Journal	Biological chemistry Hoppe-Seyler
Issue Number	2
Volume Number	371
Language	English
Publisher	Walter de Gruyter GmbH
Publisher Date	1990-01-01
Access Restriction	Open
Subject Keyword	Biological Chemistry Hoppe-seyler Analytical Chemistry Antibody Development Salt Dna Elisa Digoxigenin Sensitive Labeled Labeling and Detection Journal: Biological chemistry Hoppe-Seyler, Issue- 5
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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