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. 1989 Jul 25;17(14):5587–5595. doi: 10.1093/nar/17.14.5587

Highly sensitive detection of DNA using enzyme-linked DNA-probe. 1. Colorimetric and fluorometric detection.

A Murakami 1, J Tada 1, K Yamagata 1, J Takano 1
PMCID: PMC318181  PMID: 2762149

Abstract

In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide was synthesized. Oligonucleotide complementary to M13mp8 phage DNA was linked to alkaline phosphatase via a crosslinker and a spacer. M13mp8 phage DNA (single strand) immobilized on the nitrocellulose membrane was hybridized with the enzyme-linked oligonucleotide. The hybrid was detected with three detection methods; (1)colorimetric detection in solution, (2)colorimetric one on membranes, and (3)fluorometric one in solution. Methods(2) and (3) gave high sensitivities to detect as low as several to several tens attomoles of DNA and it was found that those methods with enzyme-linked oligonucleotides are potent for DNA-probe methodology from the viewpoint of automation.

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Selected References

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  1. Carlsson J., Drevin H., Axén R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem J. 1978 Sep 1;173(3):723–737. doi: 10.1042/bj1730723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chu B. C., Kramer F. R., Orgel L. E. Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res. 1986 Jul 25;14(14):5591–5603. doi: 10.1093/nar/14.14.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chu B. C., Kramer F. R., Orgel L. E. Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res. 1986 Jul 25;14(14):5591–5603. doi: 10.1093/nar/14.14.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu B. C., Wahl G. M., Orgel L. E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 1983 Sep 24;11(18):6513–6529. doi: 10.1093/nar/11.18.6513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grassetti D. R., Murray J. F., Jr Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys. 1967 Mar;119(1):41–49. doi: 10.1016/0003-9861(67)90426-2. [DOI] [PubMed] [Google Scholar]
  6. Hopman A. H., Wiegant J., van Duijn P. A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. II. Effects of variations in ligand structure on the in situ detection of mercurated probes. Histochemistry. 1986;84(2):179–185. doi: 10.1007/BF00499830. [DOI] [PubMed] [Google Scholar]
  7. Jablonski E., Moomaw E. W., Tullis R. H., Ruth J. L. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986 Aug 11;14(15):6115–6128. doi: 10.1093/nar/14.15.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Li P., Medon P. P., Skingle D. C., Lanser J. A., Symons R. H. Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens. Nucleic Acids Res. 1987 Jul 10;15(13):5275–5287. doi: 10.1093/nar/15.13.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Suresh M. R., Milstein C. A direct antigen-binding assay to screen hybridoma supernatants. Anal Biochem. 1985 Nov 15;151(1):192–195. doi: 10.1016/0003-2697(85)90071-5. [DOI] [PubMed] [Google Scholar]
  12. Vincent C., Tchen P., Cohen-Solal M., Kourilsky P. Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses. Nucleic Acids Res. 1982 Nov 11;10(21):6787–6796. doi: 10.1093/nar/10.21.6787. [DOI] [PMC free article] [PubMed] [Google Scholar]

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