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Preparation of Direct, Enzyme-Labeled DNA Probes

  • Ian Durrant
  • Timothy Stone
Protocol
  • 121 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The direct labeling of nucleic acid probes with horseradish peroxidase (HRP) was first described by Renz at European Molecular Biology Laboratory (EMBL) in 1984 (1). The methodology was combined with enhanced chemiluminescence (2) (a light-producing HRP catalyzed reaction based on luminol) (see  Chapter 26) allowing the detection of specific hybrids on membranes (3). Further development led to the availability of the first light-based nucleic acid detection system; this was capable of detecting 2.5 pg of target nucleic acid on genomic Southern blots (4). Subsequent protocol and reagent improvements now enable researchers to reliably detect 0.5 pg of target nucleic acid. Recent advances have now elucidated an analogous system for the labeling of DNA probes with alkaline phosphatase (AP), offering even higher sensitivity when combined with the new dioxetane based chemiluminescence substrates (see  Chapter 26).

Keywords

Boiling Water Bath Yeast Artificial Chromosome Specific Hybrid Label Reagent Direct Label 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Renz, M. and Kurz, C. (1984) A colorimetric method for DNA hybridization. Nucleic Acids Res. 12, 3435–3444.PubMedCrossRefGoogle Scholar
  2. 2.
    Whitehead, T. P., Thorpe, G. H. G., Carter, C. J. N., Groucutt, C, and Kricka, L. J. (1983) Enhanced chemiluminescence procedure for sensitive determination of peroxidaselabelled conjugates in immunoassay. Nature 305, 158–159.CrossRefGoogle Scholar
  3. 3.
    Pollard-Knight, D., Read, C. A., Downes, M. J., Howard, L. A., Leadbetter, M. R., Pheby, S. A., McNaughton, E., Syms, A., and Brady, M. A. W. (1990) Non-radioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase. Anal. Biochem. 185, 84–89.PubMedCrossRefGoogle Scholar
  4. 4.
    Durrant, I. (1990) Light based detection of biomolecules. Nature 346, 297–298.PubMedCrossRefGoogle Scholar
  5. 5.
    Durrant, I., Benge, L. C. A., Sturrock, C., Devenish, A. T., Howe, R., Roe, S., Moore, M., Scozzafava, G., Proudfoot, L. M. F., Richardson, T. C., and McFarthing, K. G. (1990) The application of enhanced chemiluminescence to membrane based nucleic acid detection. Biotechniques 8, 564–570.PubMedGoogle Scholar
  6. 6.
    Stone, T. and Durrant, I. (1991) Enhanced chemiluminescence for the detection of membrane bound nucleic acid sequences. Genet. Anal.: Tech. Appl. 8, 230–237.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Ian Durrant
    • 1
  • Timothy Stone
    • 1
  1. 1.Research and DevelopmentAmersham Pharmacia Biotech, Ltd.UK

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