Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Nov 11;19(21):6015–6019. doi: 10.1093/nar/19.21.6015

Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.

T K Christopoulos 1, E P Diamandis 1, G Wilson 1
PMCID: PMC329060  PMID: 1719486

Abstract

We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.

Full text

PDF
6015

Images in this article

6018Image
on p.6018
6018Image
on p.6018
6018Image
on p.6018

Selected References

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

  1. Bronstein I., McGrath P. Chemiluminescence lights up. Nature. 1989 Apr 13;338(6216):599–600. doi: 10.1038/338599a0. [DOI] [PubMed] [Google Scholar]
  2. Christopoulos T. K., Lianidou E. S., Diamandis E. P. Ultrasensitive time-resolved fluorescence method for alpha-fetoprotein. Clin Chem. 1990 Aug;36(8 Pt 1):1497–1502. [PubMed] [Google Scholar]
  3. Dahlén P. Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time-resolved fluorometry. Anal Biochem. 1987 Jul;164(1):78–83. doi: 10.1016/0003-2697(87)90370-8. [DOI] [PubMed] [Google Scholar]
  4. Diamandis E. P. Analytical methodology for immunoassays and DNA hybridization assays--current status and selected systems--critical review. Clin Chim Acta. 1990 Dec 17;194(1):19–50. doi: 10.1016/0009-8981(90)90302-9. [DOI] [PubMed] [Google Scholar]
  5. Diamandis E. P. Detection techniques for immunoassay and DNA probing applications. Clin Biochem. 1990 Oct;23(5):437–443. doi: 10.1016/0009-9120(90)90221-f. [DOI] [PubMed] [Google Scholar]
  6. Diamandis E. P. Immunoassays with time-resolved fluorescence spectroscopy: principles and applications. Clin Biochem. 1988 Jun;21(3):139–150. doi: 10.1016/0009-9120(88)90001-x. [DOI] [PubMed] [Google Scholar]
  7. Diamandis E. P., Morton R. C., Reichstein E., Khosravi M. J. Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays. Anal Chem. 1989 Jan 1;61(1):48–53. doi: 10.1021/ac00176a011. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Kahan I., Papanastasiou-Diamandi A., Ellis G., Makela S. K., McLaurin J., D'Costa M., Diamandis E. P. Sensitive time-resolved fluorescence immunoassay of somatotropin in serum. Clin Chem. 1990 Mar;36(3):503–508. [PubMed] [Google Scholar]
  10. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  11. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–273. doi: 10.1101/sqb.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  12. Oser A., Collasius M., Valet G. Multiple end labeling of oligonucleotides with terbium chelate-substituted psoralen for time-resolved fluorescence detection. Anal Biochem. 1990 Dec;191(2):295–301. doi: 10.1016/0003-2697(90)90222-u. [DOI] [PubMed] [Google Scholar]
  13. Pollard-Knight D., Simmonds A. C., Schaap A. P., Akhavan H., Brady M. A. Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence. Anal Biochem. 1990 Mar;185(2):353–358. doi: 10.1016/0003-2697(90)90307-u. [DOI] [PubMed] [Google Scholar]
  14. Syvänen A. C., Tchen P., Ranki M., Söderlund H. Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids. Nucleic Acids Res. 1986 Jan 24;14(2):1017–1028. doi: 10.1093/nar/14.2.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES