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. 1990 Jun 11;18(11):3175–3183. doi: 10.1093/nar/18.11.3175

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

S Sauvaigo 1, B Fouqué 1, A Roget 1, T Livache 1, H Bazin 1, C Chypre 1, R Téoule 1
PMCID: PMC330921  PMID: 2162518

Abstract

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIV1 in extracted DNA.

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Selected References

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

  1. Beck B. N., Ho S. N. Increased specificity of PCR-amplified products by size-fractionation of restriction enzyme-digested template genomic DNA. Nucleic Acids Res. 1988 Sep 26;16(18):9051–9051. doi: 10.1093/nar/16.18.9051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chehab F. F., Kan Y. W. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9178–9182. doi: 10.1073/pnas.86.23.9178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Commerford S. L. Iodination of nucleic acids in vitro. Biochemistry. 1971 May 25;10(11):1993–2000. doi: 10.1021/bi00787a005. [DOI] [PubMed] [Google Scholar]
  4. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goldkorn T., Prockop D. J. A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes. Nucleic Acids Res. 1986 Nov 25;14(22):9171–9191. doi: 10.1093/nar/14.22.9171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kemp D. J., Smith D. B., Foote S. J., Samaras N., Peterson M. G. Colorimetric detection of specific DNA segments amplified by polymerase chain reactions. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2423–2427. doi: 10.1073/pnas.86.7.2423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Manning J., Pellegrini M., Davidson N. A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes. Biochemistry. 1977 Apr 5;16(7):1364–1370. doi: 10.1021/bi00626a020. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Ou C. Y., Kwok S., Mitchell S. W., Mack D. H., Sninsky J. J., Krebs J. W., Feorino P., Warfield D., Schochetman G. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988 Jan 15;239(4837):295–297. doi: 10.1126/science.3336784. [DOI] [PubMed] [Google Scholar]
  10. Roget A., Bazin H., Teoule R. Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl. Nucleic Acids Res. 1989 Oct 11;17(19):7643–7651. doi: 10.1093/nar/17.19.7643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  12. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  13. Saiki R. K., Walsh P. S., Levenson C. H., Erlich H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. doi: 10.1073/pnas.86.16.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  15. Schulhof J. C., Molko D., Teoule R. The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups. Nucleic Acids Res. 1987 Jan 26;15(2):397–416. doi: 10.1093/nar/15.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schwab M., Alitalo K., Varmus H. E., Bishop J. M., George D. A cellular oncogene (c-Ki-ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells. Nature. 1983 Jun 9;303(5917):497–501. doi: 10.1038/303497a0. [DOI] [PubMed] [Google Scholar]
  17. Simmonds P., Balfe P., Peutherer J. F., Ludlam C. A., Bishop J. O., Brown A. J. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol. 1990 Feb;64(2):864–872. doi: 10.1128/jvi.64.2.864-872.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  19. Syvänen A. C., Bengtström M., Tenhunen J., Söderlund H. Quantification of polymerase chain reaction products by affinity-based hybrid collection. Nucleic Acids Res. 1988 Dec 9;16(23):11327–11338. doi: 10.1093/nar/16.23.11327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Syvänen A. C., Laaksonen M., Söderlund H. Fast quantification of nucleic acid hybrids by affinity-based hybrid collection. Nucleic Acids Res. 1986 Jun 25;14(12):5037–5048. doi: 10.1093/nar/14.12.5037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vosberg H. P. The polymerase chain reaction: an improved method for the analysis of nucleic acids. Hum Genet. 1989 Aug;83(1):1–15. doi: 10.1007/BF00274139. [DOI] [PubMed] [Google Scholar]
  22. Wilchek M., Bayer E. A. The avidin-biotin complex in bioanalytical applications. Anal Biochem. 1988 May 15;171(1):1–32. doi: 10.1016/0003-2697(88)90120-0. [DOI] [PubMed] [Google Scholar]

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