Using DNA Fragments as Probes

William M. Strauss1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.3
DOI:  10.1002/0471142727.mb0603s13
Online Posting Date:  May, 2001
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Abstract

All hybridization methods depend upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their Tm (melting temperature). A detailed procedure is described in which bacteriophage plaques or bacterial colonies bound to a filter membrane are detected by hybridization with a radioactive probe. Hybridization proceeds on prewet filters placed in a sealable plastic bag. After hybridization the filters are removed from the sealed bag, excess probe is washed off, and the filters are autoradiographed to identify the clones that have hybridized with the probe. An differs mainly in that formamide is not used in the hybridization solution.

     
 
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Table of Contents

  • Section II: Hybridization with Radioactive Probes
  • Basic Protocol 1: Hybridization in Formamide
  • Alternate Protocol 1: Hybridization in Aqueous Solution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Hybridization in Formamide

  Materials
  • Nitrocellulose membrane filters bearing plaques, colonies, or DNA (units 6.1 & 6.2)
  • recipeHybridization solution I
  • Radiolabeled probe, 1 to 15 ng/ml (unit 3.5)
  • recipe2 mg/ml sonicated herring sperm DNA
  • recipeHigh‐stringency wash buffer I
  • recipeLow‐stringency wash buffer I
  • Sealable bags
  • 42°C incubator
  • Water bath adjusted to washing temperature (see commentary)
  • Glass baking dish
  • Additional reagents and equipment for autoradiography ( appendix 3A)

Alternate Protocol 1: Hybridization in Aqueous Solution

  Additional Materials
  • recipeHybridization solution II
  • recipeLow‐stringency wash buffer II
  • recipeHigh‐stringency wash buffer II
  • 65°C incubator
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Figures

Videos

Literature Cited

Literature Cited
   Benton, W.D. and Davis, R.W. 1977. Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196:180.
   Botchan, M., Topp, W., and Sambrook, J. 1976. The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell 9:269‐287.
   Church, G. and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991‐1995.
   Denhardt, D. 1966. A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641‐646.
   Gillespie, D. and Spiegelman, S. 1965. A quantitative assay for DNA–RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12:829‐842.
   Grunstein, M. and Hogness, D. 1975. Colony Hybridization: A method for the isolating of cloned DNA's that contain a specific gene. Proc. Natl. Acad. Sci. U.S.A. 72:3961.
   Jeffreys, A.J. and Flavell, R.J. 1977. A physical map of the DNA region flanking the rabbit β globin gene. Cell 12:429‐439.
   Southern, E.M. 1975. Detection of specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
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