Abstract
The recombinant Ca2+-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10−15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.
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Abbreviations
- BCIP:
-
5-bromo-4-chlorine-3-indolylphosphate
- HCV:
-
hepatitis C virus
- DMEG:
-
ethylene glycol dimethacryl ester
- NPP:
-
p-nitrophenylphosphate
- PCR:
-
polymerase chain reaction
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Original Russian Text © V.V. Borisova, I.A. Pyshnaya, D.V. Pyshnyi, L.A. Frank, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 6, pp. 792–798.
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Borisova, V.V., Pyshnaya, I.A., Pyshnyi, D.V. et al. A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter. Russ J Bioorg Chem 34, 709–715 (2008). https://doi.org/10.1134/S1068162008060101
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DOI: https://doi.org/10.1134/S1068162008060101