Skip to main content
SpringerLink
Log in

Labeling and Detection of Nucleic Acids

  • Chapter
Nonradioactive Analysis of Biomolecules

Part of the book series: Springer Lab Manuals ((SLM))

  • 1956 Accesses

Abstract

The highly sensitive non-radioactive DIG system allows specific detection Principle and of 0.01-0.03 pg DNA or RNA within 1-16 h with colorimetric BCIP/NBT applications detection (McGadey, 1970) or 15-30 min with chemiluminescent detection [Disodium 3- (4-methoxyspiro-{1,2 - dioxetane-3,2’-(5’-chloro) tricyclo [3.3.1.1 3’7]decan}-4-yl)phenyl phosphate (CSPD) or Disodium 2-chloro5-(4-methoxyspiro {1,2-dioxetane-3,2’-(5’-chloro)tricyclo [3.3.1.13’7] decan}-4-y1)-1-phenyl phosphate (CDP-Star) (Bronstein et al., 1989)] in dot-, slot-, Southern and Northern blots, avoiding any significant background on nylon membranes (Kessler et al., 1990; Höltke et al., 1990; Seibl et al., 1990; Mühlegger et al., 1990; Kessler, 1991; Höltke, 1995). Nitrocellulose membranes in combination with chemiluminescence substrates are less sensitive and require the addition of an enhancer [e.g., NitroBlock, from Tropix, Inc. (Höltke, 1995)].

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  • Arnold N, Seibl R, Kessler C, Wienberg J (1992) Non-radioactive in situ hybridization with digoxigenin-labeled DNA probes. Biotechnic & Histochemistry 67 (1992) 59–67.

    Article  CAS  Google Scholar 

  • Baldino F, Jr, Robbins E, Grega D, Meyers SL, Springer, JE, Lewis ME (1989) Non-radioactive detection of NGF-receptor mRNA with digoxigenin-UTP labeled RNA probes. Neurosci Abstr 15:864.

    Google Scholar 

  • Bronstein I, Cate RL, Lazzari K, Ramachandram KL, Voyta JC (1989) Chmiluminescent 1,2-dioxetane based enzyme substrates and their application in the detection of DNA. Photochem Photobiol 49:9.

    Google Scholar 

  • Casacuberta JM, Jardi R, Buti M, Puigdomenech P, SanSegundo B (1988) Comparison of different non-isotopic methods for hepatitis-B virus detection in human serum. Nucleic Acids Res 16:11834.

    Article  PubMed  CAS  Google Scholar 

  • Cohen SM (1990) Specification of limb development in the Drosophila embryo by positional cues from segmentation genes. Nature (London) 343:173–177.

    Article  CAS  Google Scholar 

  • Dooley S, Radtke J, Blin N, Unteregger G (1988) Rapid detection of DNA-binding factors using protein-blotting and digoxigenin-dUTP marked probes. Nucleic Acids Res 16:11829.

    Article  CAS  Google Scholar 

  • Dorward DW, Garon CF (1989) DNA-binding proteins-in cells and membrane plots of Neisseria gonorrhoeae. J Bacteriol 171:4196–4201.

    PubMed  CAS  Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13.

    Article  PubMed  CAS  Google Scholar 

  • Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endo-nuclease fragments to high specific activity. Anal Biochem 137:266–267.

    Article  PubMed  CAS  Google Scholar 

  • Gebeyehu G, Rao PY, SooChan P, Simms DA, Kievan L (1987) Novel biotinylated nu-cleotide analogue for labeling and colorimetric detection of DNA. Nucleic Acids Res 15:4513–4534.

    Article  PubMed  CAS  Google Scholar 

  • Grega DS, Cavanagh TJ, Grimme S, Martin R, Lewis M, Robbins E, Robbins E, Baldino F Jr (1989) Localization of neuronal mRNA by in situ hybridization using a non-radioactive detection method. Neurosci Abstr 15:739.

    Google Scholar 

  • Grega DS, Cavanagh TJ, Martin R, Lewis M, Baldino F Jr (1989) in situ hybridization histochemistry using a new non-radioactive detection method. Advances in Gene Technology: Molecular Neurobiology and Neuropharmacology, ICSU Short Reports 9:69.

    Google Scholar 

  • Herrington CS, Burns J, Graham AK, Bhatt B, McGee, JD (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. II. Simultaneous detection of human and papilloma virus nucleic acids in individual nuclei. J Clin Pathol (Lond) 42:601–606.

    Article  CAS  Google Scholar 

  • Herrington CS, Burns J, Graham AK, Evans M, McGee JD (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. I: Relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells. J Clin Pathol (Lond) 42:592–600.

    Article  CAS  Google Scholar 

  • Höltke HJ, Ankenbauer W, Mühlegger K, Rein R, Sagner G, Seibl R, Walter T (1995) The digoxigenin (Dig) system for non-radioactive labeling and detection of nucleic acids - an overview. Cell Mol Biol 41: 883–905.

    PubMed  Google Scholar 

  • Höltke HJ, Kessler C (1990) Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res 18:5843–5851.

    Article  PubMed  Google Scholar 

  • Höltke HJ, Sagner G, Kessler C, Schmitz GG (1992) Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications. Biotechniques 12:104–113.

    PubMed  Google Scholar 

  • Höltke HJ, Seibl R, Burg J, Mühlegger K, Kessler C (1990) Non-radioactive labeling and detection of nucleic acids: II. Optimization of the digoxigenin system. Biol Chem Hoppe-Seyler 371:929–938.

    Article  PubMed  Google Scholar 

  • Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR protocols. Academic Press Inc., San Diego, CA.

    Google Scholar 

  • Jessberger R, Berg P (1991) Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system. Mol Cell Biol 11:445–457.

    PubMed  CAS  Google Scholar 

  • Kessler C (1990) Detection of nucleic acids by enzyme-linked immunosorbent assay (ELISA) technique: An example for the development of a novel non-radioactive labeling and detection system with high sensitivity. In: Obe G (ed) Advances in Mutagenesis Research, Springer-Verlag, Berlin/Heidelberg, pp 105–152.

    Chapter  Google Scholar 

  • Kessler C (1991) The digoxigenin:anti-digoxigenin (DIG) technology - a survey on the concept and realization of a novel bioanalytical indicator system. Mol Cell Probes 5:161–205.

    Article  PubMed  CAS  Google Scholar 

  • Kessler C, Höltke H-J, Seibl R, Burg J, Mühlegger K (1990) 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). Biol Chem Hoppe-Seyler 371:917–927.

    Article  PubMed  CAS  Google Scholar 

  • Kessler C, Sagner G, Höltke H-J (1991) Non-radioactive dideoxy sequencing using the digoxigenin system. The 1991 San Diego Conference of Nucleic Acids, San Diego.

    Google Scholar 

  • Kimpton CP, Corbitt G, Morris, DJ (1989) Detection of cytomegalovirus DNA using probes labeled with digoxigenin. J Virol Methods 24:335–346.

    Article  PubMed  CAS  Google Scholar 

  • Krieg PA, Melton DA (1987) in vitro RNA synthesis with SP6 RNA polymerase. Methods Enyzmol 155:397–415.

    Article  CAS  Google Scholar 

  • Lahaye T, Rüger B, Toepsch S, Thalhammer J, Schulze-Lefert P (1986) Detection of single-copy sequences with digoxigenin-labeled probes in a complex plant genome after separation on pulsed field gels. Biotechniques 21:1067–1072.

    Google Scholar 

  • Lichter P, Tang C-JC, Call K, Hermanson G, Evans GA, Housman D, Ward, DC (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64–69.

    Article  PubMed  CAS  Google Scholar 

  • Lion T, Haas OA (1990) Nonradioactive labelling of probe with digoxigenin by polymer-ase chain reaction. Anal Biochem 188: 335–337.

    Article  PubMed  CAS  Google Scholar 

  • Manstein DJ, Titus MA, De Lozanne A, Spudich JA (1989) Gene replacement in Dictyostelium: generation of myosin null mutant. EMBO J 8:923–932.

    PubMed  CAS  Google Scholar 

  • McGadey J (1990) Tetrazolium method for nunspecific alkaline phosphatase. Histochemie 23: 180–184.

    Google Scholar 

  • Mühlegger K, Huber E, von der Eltz H, Rüger R, Kessler C (1990) Non-radioactive labeling and detection of nucleic acids: IV. Synthesis and properties of the nucleotide corn-pounds of the digoxigenin system and of photodigoxigenin. Biol Chem Hoppe-Seyler 371:953–965.

    Article  PubMed  Google Scholar 

  • Nakano Y, Guerrero I, Hidalgo A, Taylor A, Whittle JRS, Ingham PW (1989) A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched. Nature (London) 341:508–513.

    Article  CAS  Google Scholar 

  • Persing DH, Smith TF, Tenover FC, White TJ (1993) Diagnostic Molecular Microbiology - Principles and Applications. American Society for Microbiology, Washington, DC.

    Google Scholar 

  • Reischl U, Rüger R, Kessler C (1992) Nonradioactive labeling of PCR products. In: White BA (ed) PCR: Selected Protocols and Applications. The Humana Press, Clifton, NJ, pp 51–62.

    Google Scholar 

  • Roychoudbury R, Wu R (1980) Terminal transferase-catalyzed addition of nucleotides to the 3’ termini of DNA. Methods Enzymol 65:43–62.

    Article  Google Scholar 

  • Rüger B, Thalhammer J, Obermaier J, Gruenewald-Janho S (1996) Experimental procedure for the detection of a rare human mRNA with the DIG-system. Biochemica 3:96.

    Google Scholar 

  • Rüger R, Höltke HJ, Sagner G, Seibl R, Kessler C (1990) Rapid labeling methods using the DIG-system: incorporation of digoxigenin in PC reactions and labeling of nucleic acids with photodigoxigenin. Fresenius’ Zeitschrift für Analytische Chemie 337:114.

    Google Scholar 

  • Schäfer R, Zischler, H, Epplen, JT (1988) DNA fingerprinting using non-radioactive oligonucleotide probes specific for simple repeats. Nucleic Acids Res 16:9344.

    Article  PubMed  Google Scholar 

  • Schmitz GG, Walter T, Seibl R, Kessler C (1991) Non-radioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem 192:222–231.

    Article  PubMed  CAS  Google Scholar 

  • Seibl R, Höltke H-J, Rüger R, Meindl A, Zachau HG, Raßhofer R, Roggendorf M, Wolf H, Arnold N, Wienberg J, Kessler C (1990) Non-radioactive labeling and detection of nucleic acids: III. Applications of the digoxigenin system. Biol Chem Hoppe-Seyler 371:939–951.

    Article  PubMed  CAS  Google Scholar 

  • Suske G, Gross B, Beato M (1989) Non-radioactive method to visualize specific DNA-protein interactions in the band shift assay. Nucleic Acids Res 17:4405.

    Article  PubMed  CAS  Google Scholar 

  • Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81–85.

    Article  PubMed  CAS  Google Scholar 

  • West S, Schröder J, Kunz W (1990) A multiple-staining procedure for the detection of different DNA fragments on a single blot. Anal Biochem 190:254–258.

    Article  PubMed  CAS  Google Scholar 

  • Young WS, 111 (1989) Simultaneous use of digoxigenin-and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides (Edinburgh) 12:271–275.

    Article  Google Scholar 

  • Zischler H, Panda I, Schäfer R, Schmid M, Epplen JT (1989) Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet 82:227–233.

    Article  PubMed  CAS  Google Scholar 

  • Zischler H, Schäfer R, Epplen JT (1989) Non-radioactive oligonucleotide fingerprinting in the gel. Nucleic Acids Res 17:4411.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Roche Diagnostics GmbH, Roche Molecular Biochemicals, Nonnenwald, 2, Penzberg, 82377, Germany

    Hans-Joachim Höltke, Kelly King, Cathy Hoover, Irmgard Obermaier, Barbara RÜger, Klaus MÜhlegger & Christoph Kessler

Authors
  1. Hans-Joachim Höltke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kelly King
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cathy Hoover
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Irmgard Obermaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barbara RÜger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Klaus MÜhlegger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christoph Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Roche Molecular Systems Werk Tutzing, Roche Diagnostics GmbH, Bahnhofstrasse 9-15, 82327, Tutzing, Germany

    Christoph Kessler

  2. Gene Center/Institute of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377, München, Germany

    Christoph Kessler

Rights and permissions

Reprints and permissions

Copyright information

© 2000 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Höltke, HJ. et al. (2000). Labeling and Detection of Nucleic Acids. In: Kessler, C. (eds) Nonradioactive Analysis of Biomolecules. Springer Lab Manuals. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-57206-7_4

Download citation

  • DOI: https://doi.org/10.1007/978-3-642-57206-7_4

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-540-64601-3

  • Online ISBN: 978-3-642-57206-7

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation