Abstract
The labeling of long nucleic acid probes is based on the work first described by Renz at the European Molecular Biology Laboratories (EMBL) (Renz and Kurz, 1984). Single-stranded nucleic acid (RNA or DNA) is labeled with a positively charged, modified, horseradish peroxidase (HRP) complex in a rapid, reliable, and simple reaction process to produce a stable probe. In conjunction with enhanced chemiluminescence (ECL), a light-based detection system, the HRP-labeled probes can be used to detect single copy genes in as little as 1 µg of a restriction enzyme digest of human DNA blotted onto either nylon or nitrocellulose membranes (Stone and Durrant, 1991). The light output of the ECL reaction is captured on X-ray film; for most high sensitivity applications the exposure time required is less than 60 min, although up to 4 h exposures are possible. For high target applications the associated rapid hybridization and rapid detection procedures enable the whole process to be completed in 1 working day.
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References
Amersham International (1990) Detection of nucleic acids and protein with light: ECL technical manual
Boniszewski ZAM, Comley JS, Hughes B, Read CA (1990) The use of charge-coupled devices in the quantitative evaluation of images, on photographic film or membranes, obtained following electrophoretic separation of DNA fragments. Electrophoresis 11: 432–440
Connolly BA, Rider P (1985) Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes. Nucleic Acids Res 13: 4485–4502
Cunningham M (1991) Nucleic acid detection with light. Life Science 6: 2–5
Cunningham M, Harvey B, Benge L, Wheeler C (1991) ECL random prime system: protocol variations. Highlights 2: 9–10
Durrant I (1990) Light based detection of biomolecules. Nature (London) 346: 297–298
Durrant I, Benge LCA, Sturrock C, Devenish AT, Howe R, Roe S, Moore M, Scozzafava G, Proudfoot LMF, Richardson TC, McFarthing KG (1990) The application of enhanced chemiluminescence to membrane-based nucleic acid detection. Bio Techniques 8: 564–570
Evans MR, Benge LCA, Devenish AT, Durrant I, Fowler SJ, Harding ER, Howe R, Richardson TC, Scozzafava G, Sturrock C, Proudfoot LMF (1990) Chemiluminescence: nucleic acid detection for the future. In Proceedings of the 6th International Congress on Rapid Methods and Automation in Microbiology and Immunology, Helsinki
Fowler SJ, Harding ER, Evans MR (1990) Labeling of oligonucleotides with horseradish peroxidase and detection using enhanced chemiluminescence. Technique 2: 261–267
Grimsley G, Witt C, Saveracker G, Dawkins RL (1991) HLA DRB typing of polymerase chain reaction (PCR) products using horseradish peroxidase labeled sequence specific oligonucleotides ( SSOs) and enhanced chemiluminescence. Highlights 3: 1–3
Hawkins E, Cumming R (1990) Enhanced chemiluminescence for tissue antigen and cellular viral DNA detection. J Histochem Cytochem 38: 415–419
Hutton JR (1977) Renaturation kinetics and thermal stability of DNA in aqueous solu` tions of formamide and urea. Nucleic Acids Res 4: 3537–3555
Lambalk JJM (1991) ECL direct system: single copy gene detection in tomato plants. Highlights 2: 3–4
Leong MM, Fox GR (1988) Enhancement of luminol-based immunodot and western blotting assays by iodophenol. Analyt Biochem 172: 145–150
Misiura K, Durrant I, Evans MR, Gait MJ (1990) Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides. Nucleic Acids Res 18: 4345–4354
Moore M (1991) ECL direct system: stringency washes without the use of urea in a northern blotting application. Highlights 2: 5–6
Pollard-Knight D (1990) Current methods in nonradioactive nucleic acid labeling and detection. Technique 2: 113–132
Pollard-Knight D, Read CA, Downes MJ, Howard LA, Leadbetter MR, Pheby SA, McNaughton E, Syms A, Brady MAW (1990) Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase. Analyt Biochem 185: 84–89
Renz M, Kurz C (1984) A colorimetric method for DNA hybridization. Nucleic Acids Res 12: 3435–3444
Simmonds AC, Cunningham M, Durrant I, Fowler SJ, Evans MR (1991) Enhanced chemiluminescence in filter-based DNA detection. Clinical Chemistry 37: 1527–1528
Sorg R, Enczmann J, Sorg U, Kogler G, Schneider EM, Wernet P (1990) Specific nonradioactive detection of PCR-amplified HIV-sequences with enhanced chemiluminescence labeling ( ECL) — an alternative to conventional hybridization with radioactive isotopes. Life Science 2: 3–4
Stone T, Durrant I (1992) Enhanced chemiluminescence for the detection of membrane bound nucleic acid sequences. Genetic Analysis: Techniques and Applications 8: 230–237
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© 1992 Springer-Verlag Berlin Heidelberg
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Durrant, I. (1992). Direct Peroxidase Labeling of Hybridization Probes and Chemiluminescence Detection. In: Kessler, C. (eds) Nonradioactive Labeling and Detection of Biomolecules. Springer Laboratory. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-00144-8_8
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DOI: https://doi.org/10.1007/978-3-662-00144-8_8
Publisher Name: Springer, Berlin, Heidelberg
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