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. 1994 Jul 11;22(13):2604–2611. doi: 10.1093/nar/22.13.2604

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

M Kwiatkowski 1, M Samiotaki 1, U Lamminmäki 1, V M Mukkala 1, U Landegren 1
PMCID: PMC308216  PMID: 8041624

Abstract

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.

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Selected References

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  1. Cardullo R. A., Agrawal S., Flores C., Zamecnik P. C., Wolf D. E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carrell R. W., Jeppsson J. O., Laurell C. B., Brennan S. O., Owen M. C., Vaughan L., Boswell D. R. Structure and variation of human alpha 1-antitrypsin. Nature. 1982 Jul 22;298(5872):329–334. doi: 10.1038/298329a0. [DOI] [PubMed] [Google Scholar]
  3. Chang P. K. Iodination of 2'-deoxycytidine and related substances. A reinvestigation of the structures of the by-product, C9H10I2N2O5, and its derivatives. J Org Chem. 1965 Nov;30(11):3913–3915. doi: 10.1021/jo01022a076. [DOI] [PubMed] [Google Scholar]
  4. Diamandis E. P., Christopoulos T. K. Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays. Anal Chem. 1990 Nov 15;62(22):1149A–1157A. doi: 10.1021/ac00221a001. [DOI] [PubMed] [Google Scholar]
  5. Haralambidis J., Angus K., Pownall S., Duncan L., Chai M., Tregear G. W. The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels. Nucleic Acids Res. 1990 Feb 11;18(3):501–505. doi: 10.1093/nar/18.3.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Haralambidis J., Chai M., Tregear G. W. Preparation of base-modified nucleosides suitable for non-radioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jun 25;15(12):4857–4876. doi: 10.1093/nar/15.12.4857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Hemmilä I., Mukkala V. M., Latva M., Kiilholma P. Di- and tetracarboxylate derivatives of pyridines, bipyridines and terpyridines as luminogenic reagents for time-resolved fluorometric determination of terbium and dysprosium. J Biochem Biophys Methods. 1993 Jul;26(4):283–290. doi: 10.1016/0165-022x(93)90029-n. [DOI] [PubMed] [Google Scholar]
  9. Iitiä A., Høgdall E., Dahlen P., Hurskainen P., Vuust J., Siitari H. Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry. PCR Methods Appl. 1992 Nov;2(2):157–162. doi: 10.1101/gr.2.2.157. [DOI] [PubMed] [Google Scholar]
  10. Landegren U., Kaiser R., Sanders J., Hood L. A ligase-mediated gene detection technique. Science. 1988 Aug 26;241(4869):1077–1080. doi: 10.1126/science.3413476. [DOI] [PubMed] [Google Scholar]
  11. Palmieri G., Romano G., Casamassimi A., D'Urso M., Little R. D., Abidi F. E., Schlessinger D., Lagerström M., Malmgren H., Steen-Bondeson M. L. 1.5-Mb YAC contig in Xq28 formatted with sequence-tagged sites and including a region unstable in the clones. Genomics. 1993 Jun;16(3):586–592. doi: 10.1006/geno.1993.1234. [DOI] [PubMed] [Google Scholar]
  12. Parik J., Kwiatkowski M., Lagerkvist A., Lagerström Fermér M., Samiotaki M., Stewart J., Glad G., Mendel-Hartvig M., Landegren U. A manifold support for molecular genetic reactions. Anal Biochem. 1993 May 15;211(1):144–150. doi: 10.1006/abio.1993.1245. [DOI] [PubMed] [Google Scholar]

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