Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Nov 25;13(22):8083–8091. doi: 10.1093/nar/13.22.8083

Detection of sub-picogram quantities of specific DNA sequences on blot hybridization with biotinylated probes.

V T Chan, K A Fleming, J O McGee
PMCID: PMC322111  PMID: 2933635

Abstract

A sensitive method for detecting biotinylated DNA probes on dot and Southern blots is described which is based on the principle outlined by Leary et al (1). This system has two main components: detection of biotinylated DNA by a two-step procedure with streptavidin and poly(alkaline phosphatase); and blocking background with Tween 20. 32fg and 80fg of lambda phage DNA was detected on dot and Southern blot hybridizations respectively. 150fg of beta-globin was detected on Southern blots of genomic DNA. This method is fast, reproducible and can detect single copy genes in 0.25 micrograms genomic DNA on Southern blots.

Full text

PDF
8083

Images in this article

Selected References

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

  1. Bayer E. A., Wilchek M. The use of the avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal. 1980;26:1–45. doi: 10.1002/9780470110461.ch1. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Burns J., Chan V. T., Jonasson J. A., Fleming K. A., Taylor S., McGee J. O. Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells. J Clin Pathol. 1985 Oct;38(10):1085–1092. doi: 10.1136/jcp.38.10.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Gallyas F., Görcs T., Merchenthaler I. High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J Histochem Cytochem. 1982 Feb;30(2):183–184. doi: 10.1177/30.2.7061820. [DOI] [PubMed] [Google Scholar]
  7. Hsu S. M., Raine L., Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577–580. doi: 10.1177/29.4.6166661. [DOI] [PubMed] [Google Scholar]
  8. Hutchison N. J., Langer-Safer P. R., Ward D. C., Hamkalo B. A. In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold. J Cell Biol. 1982 Nov;95(2 Pt 1):609–618. doi: 10.1083/jcb.95.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaufman R. E., Kretschmer P. J., Adams J. W., Coon H. C., Anderson W. F., Nienhuis A. W. Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4229–4233. doi: 10.1073/pnas.77.7.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kempe T., Sundquist W. I., Chow F., Hu S. L. Chemical and enzymatic biotin-labeling of oligodeoxyribonucleotides. Nucleic Acids Res. 1985 Jan 11;13(1):45–57. doi: 10.1093/nar/13.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Landegent J. E., Jasen in de Wal N., Baan R. A., Hoeijmakers J. H., Van der Ploeg M. 2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp Cell Res. 1984 Jul;153(1):61–72. doi: 10.1016/0014-4827(84)90448-8. [DOI] [PubMed] [Google Scholar]
  12. Langer-Safer P. R., Levine M., Ward D. C. Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4381–4385. doi: 10.1073/pnas.79.14.4381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Manning J. E., Hershey N. D., Broker T. R., Pellegrini M., Mitchell H. K., Davidson N. A new method of in situ hybridization. Chromosoma. 1975 Nov 24;53(2):107–117. doi: 10.1007/BF00333039. [DOI] [PubMed] [Google Scholar]
  16. Miller E. C., Juhl U., Miller J. A. Nucleic acid guanine: reaction with the carcinogen N-acetoxy-2-acetylaminofluorene. Science. 1966 Sep 2;153(3740):1125–1127. doi: 10.1126/science.153.3740.1125. [DOI] [PubMed] [Google Scholar]
  17. Murasugi A., Wallace R. B. Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes. DNA. 1984 Jun;3(3):269–277. doi: 10.1089/dna.1.1984.3.269. [DOI] [PubMed] [Google Scholar]
  18. Pellegrini M., Holmes D. S., Manning J. Application of the avidin-biotin method of gene enrichment to the isolation of long double-stranded DNA containing specific gene sequences. Nucleic Acids Res. 1977 Sep;4(9):2961–2973. doi: 10.1093/nar/4.9.2961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Radford A. J. Comparisons using 35S- and 32P-labeled DNA for hybridization on nitrocellulose filters. Anal Biochem. 1983 Oct 15;134(2):269–271. doi: 10.1016/0003-2697(83)90296-8. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Shih C., Weinberg R. A. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 1982 May;29(1):161–169. doi: 10.1016/0092-8674(82)90100-3. [DOI] [PubMed] [Google Scholar]
  23. Singer R. H., Ward D. C. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7331–7335. doi: 10.1073/pnas.79.23.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES