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)

Abstract

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 

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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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