[A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter].
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 a fine-grained polymer or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with a 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 elongation of the chain using 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, ensures a high sensitivity of the assay (no less than 10(-15) mol of DNA template), and enables quantitative determination of the amount of DNA template in the tested sample.
Borisova
V V
VV
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, str. 50, Akademgorodok, Krasnoyarsk, 660036 Russia.
Pyshnaia
I A
IA
Pyshnyĭ
D V
DV
Frank
L A
LA
D053938:DNA Fragmentation; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity
D015342:DNA Probes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C010497:obelin