Direct In-Gel Hybridization of DNA With Digoxigenin-Labeled Probes

  • Saeed A. Khan
  • Mohamed S. Nawaz
Part of the Methods in Molecular Biology book series (MIMB, volume 353)


In-gel hybridization with digoxigenin (DIG)-labeled probes has been shown to detect complementary DNA sequences in dried agarose gels. Gels dried at room temperature or at 55°C in an oven do not show detectable changes in the sensitivity of detection. However, gels dried under vacuum seem to lose the sensitivity by approx 8- to 10-fold. In-gel hybridization after blotting high molecular weight T7 DNA (40 kbp) onto nylon membranes has been demonstrated to transfer the DNA to the membrane inefficiently. In-gel hybridization of DIG-labeled probes with the complementary DNA sequences has been determined to detect as little as 0.05 ng of 40-kbp T7 DNA and single copies of the erythromycin resistance marker gene ermA. Nonradioactive in-gel hybridization provides better quantitation of nucleic acids than filter hybridization in Southern and Northern blot analysis.

Key Words

Colorimetric digoxigenin direct in-gel hybridization 


  1. 1.
    Bierwerth, S., Kahl, G., Weigand, F., and Weising, K. (1992) Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic chemiluminescent detection methods. Electrophoresis 13, 115–122.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang, R. F., Cao, W.-W., and Johnson, M. G. (1991). Direct gel hybridization in place of Southern hybridization for detection of Listeria monocytogenes DNA fragments. Lett. Appl. Microbiol. 12, 224–227.PubMedCrossRefGoogle Scholar
  3. 3.
    Lueders, K. K. and Fewell, J. W. (1994) Hybridization in dried gels provides increased sensitivity compared with hybridization to blots. Biotechniques 16, 66–67.PubMedGoogle Scholar
  4. 4.
    Ahmad, I., Finklestein, J. A., and Steggles, A. W. (1990) The analysis of RNA by in situ agarose gel hybridization is more sensitive than the equivalent northern blot analysis. Biotechniques 8, 162–165.PubMedGoogle Scholar
  5. 5.
    Khan, S. A., Watson, R. H., Hayes, S. J., and Serwer, P. (1995) Specific nonproductive cleavage of packaged bacteriophage T7 DNA in vivo. Virology, 210, 409–420.PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson, D. R., and Brietenberger, C. A. (1993) In-gel hybridization to separate yeast chromosomes. Biotechniques 15, 836–837.PubMedGoogle Scholar
  7. 7.
    Zischler, H., Schafer, R., and Epplen, J. T. (1989) Non-radioactive oligonucleotide fingerprinting in the gel. Nucleic Acids Res. 17, 4411.PubMedCrossRefGoogle Scholar
  8. 8.
    Gontijo, N. F., Ribeiro, J. C., and Pena, S. D. (1990) Direct hybridization of biotin-labeled probes in dried agarose gels. Trends Genet. 6, 3.PubMedCrossRefGoogle Scholar
  9. 9.
    Yavachev, L. (1991) A rapid method of non-radioactive detection of DNA-fragments in dried agarose gels. Nucleic Acids Res. 19, 186.PubMedCrossRefGoogle Scholar
  10. 10.
    Pena, S. D., Masedo, A. M., Gontijo, M. F., Medeiros, A. M., and Ribiero, J. C. (1991) DNA bioprints: simple nonisotopic DNA fingerprints with biotinylated DNA probes. Electrophoresis 12, 146.PubMedCrossRefGoogle Scholar
  11. 11.
    Augood, S. J., Ruth, J. L., and Emerson, P. C. (1990) A rapid method of nonradioactive northern blot analysis. Nucleic Acids Res. 18, 4291.PubMedCrossRefGoogle Scholar
  12. 12.
    Gebeyehu, G., Rao, P. Y., SooChan, P., Simms, D., and Klevan, L. (1987) Novel biotinylated nucleotide analogs for labeling and colorimetric detection of DNA. Nucleic Acids Res. 15, 4513–4534.PubMedCrossRefGoogle Scholar
  13. 13.
    Höltke, H.-J., Seibl, R., Burg, J., Mühlegger, K., and Kessler, C. (1990) Nonradioactive labeling and detection of nucleic acids: II. Optimization of the digoxigenin system. Biol. Chem. Hoppe-Seyler 371, 929–938.PubMedCrossRefGoogle Scholar
  14. 14.
    Kessler, C. (1990) The digoxigenin system: principles and applications of the novel non-radioactive DNA labeling and detection system. Biotechnol. Int. 1990, 183–194.Google Scholar
  15. 15.
    Martin, R., Hoover, C., Grimme, S., Grogn, C., Höltke, J., and Kessler, C. (1990) A highly sensitive nonradioactive DNA labeling and detection system. Biotechniques 9, 762–768.PubMedGoogle Scholar
  16. 16.
    Schmitz, G. G., Walter, T., Seibl, R., and Kessler, C. (1991) Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222–231.PubMedCrossRefGoogle Scholar
  17. 17.
    Thomas, L. and Oskar, A. H. (1990) Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction. Anal. Biochem. 188, 335–337.CrossRefGoogle Scholar
  18. 18.
    Waye, J. S. and Willard, H. F. (1987) Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes. Nucleic Acids Res. 15, 7459–7569.CrossRefGoogle Scholar
  19. 19.
    Choo, K. H., Vissel, B., Nagy, A., Earle, E., and Kalitsis, P. (1991) Survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nuc. Acids Res. 19, 1179–1182.CrossRefGoogle Scholar
  20. 20.
    Lengauer, C., Speicher, M., Popp, S., et al. (1993) Chromosomal barcodes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes. Hum. Mol. Gen. 2, 505–512.PubMedCrossRefGoogle Scholar
  21. 21.
    Liebert, Y. (1990) A rapid method of non-radioactive detection of DNA-fragments in dried agarose gels. Nucleic Acids Res. 19, 186.Google Scholar
  22. 22.
    Zischler, H., Hinkkanen, A., and Studer, R. (1991) Oligonucleotide fingerprinting with (CAC) 5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci. Electrophoresis 12, 141–146.PubMedCrossRefGoogle Scholar
  23. 23.
    Khan, S. A., Nawaz, M. S., Khan, A. A., and Cerniglia, C. E. (1999) Direct in-gel hybridization of digoxigenin-labeled non-radioactive probes. Mol. Cell. Probes 13, 233–237.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Saeed A. Khan
    • 1
  • Mohamed S. Nawaz
    • 1
  1. 1.Division of Microbiology, National Center for Toxicological ResearchU.S. Food and Drug AdministrationJefferson

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