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).
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References
Renz, M. and Kurz, C. (1984) A colorimetric method for DNA hybridization. Nucleic Acids Res. 12, 3435–3444.
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.
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.
Durrant, I. (1990) Light based detection of biomolecules. Nature 346, 297–298.
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.
Stone, T. and Durrant, I. (1991) Enhanced chemiluminescence for the detection of membrane bound nucleic acid sequences. Genet. Anal.: Tech. Appl. 8, 230–237.
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© 2000 Humana Press Inc., Totowa, NJ
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Durrant, I., Stone, T. (2000). Preparation of Direct, Enzyme-Labeled DNA Probes. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:145
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DOI: https://doi.org/10.1385/1-59259-038-1:145
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
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