Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1986 Jan 24;14(2):1017–1028. doi: 10.1093/nar/14.2.1017

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

A C Syvänen, P Tchen, M Ranki, H Söderlund
PMCID: PMC339480  PMID: 3945551

Abstract

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.

Full text

PDF
1020

Images in this article

1023Image
on p.1023

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brigati D. J., Myerson D., Leary J. J., Spalholz B., Travis S. Z., Fong C. K., Hsiung G. D., Ward D. C. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology. 1983 Apr 15;126(1):32–50. doi: 10.1016/0042-6822(83)90460-9. [DOI] [PubMed] [Google Scholar]
  2. Forster A. C., McInnes J. L., Skingle D. C., Symons R. H. Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 1985 Feb 11;13(3):745–761. doi: 10.1093/nar/13.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hemmilä I., Dakubu S., Mukkala V. M., Siitari H., Lövgren T. Europium as a label in time-resolved immunofluorometric assays. Anal Biochem. 1984 Mar;137(2):335–343. doi: 10.1016/0003-2697(84)90095-2. [DOI] [PubMed] [Google Scholar]
  4. Hjelm H., Hjelm K., Sjöquist J. Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 1972 Nov 15;28(1):73–76. doi: 10.1016/0014-5793(72)80680-x. [DOI] [PubMed] [Google Scholar]
  5. Hu N., Messing J. The making of strand-specific M13 probes. Gene. 1982 Mar;17(3):271–277. doi: 10.1016/0378-1119(82)90143-3. [DOI] [PubMed] [Google Scholar]
  6. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ranki M., Palva A., Virtanen M., Laaksonen M., Söderlund H. Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples. Gene. 1983 Jan-Feb;21(1-2):77–85. doi: 10.1016/0378-1119(83)90149-x. [DOI] [PubMed] [Google Scholar]
  10. Renz M., Kurz C. A colorimetric method for DNA hybridization. Nucleic Acids Res. 1984 Apr 25;12(8):3435–3444. doi: 10.1093/nar/12.8.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Renz M. Polynucleotide-histone H1 complexes as probes for blot hybridization. EMBO J. 1983;2(6):817–822. doi: 10.1002/j.1460-2075.1983.tb01508.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  13. Sage E., Fuchs R. P., Leng M. Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. Biochemistry. 1979 Apr 3;18(7):1328–1332. doi: 10.1021/bi00574a032. [DOI] [PubMed] [Google Scholar]
  14. Soini E., Hemmilä I. Fluoroimmunoassay: present status and key problems. Clin Chem. 1979 Mar;25(3):353–361. [PubMed] [Google Scholar]
  15. Soini E., Kojola H. Time-resolved fluorometer for lanthanide chelates--a new generation of nonisotopic immunoassays. Clin Chem. 1983 Jan;29(1):65–68. [PubMed] [Google Scholar]
  16. Syvänen A. C., Alanen M., Söderlund H. A complex of single-strand binding protein and M13 DNA as hybridization probe. Nucleic Acids Res. 1985 Apr 25;13(8):2789–2802. doi: 10.1093/nar/13.8.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tchen P., Fuchs R. P., Sage E., Leng M. Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3466–3470. doi: 10.1073/pnas.81.11.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Virtanen M., Palva A., Laaksonen M., Halonen P., Söderlund H., Ranki M. Novel test for rapid viral diagnosis: detection of adenovirus in nasopharyngeal mucus aspirates by means of nucleic-acid sandwich hybridisation. Lancet. 1983 Feb 19;1(8321):381–383. doi: 10.1016/s0140-6736(83)91500-3. [DOI] [PubMed] [Google Scholar]
  19. Virtanen M., Syvänen A. C., Oram J., Söderlund H., Ranki M. Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization. J Clin Microbiol. 1984 Dec;20(6):1083–1088. doi: 10.1128/jcm.20.6.1083-1088.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES