Hybridization Analysis of DNA Blots

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.6B
DOI:  10.1002/0471142735.im1006bs06
Online Posting Date:  May, 2001
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Abstract

The principle of hybridization analysis is that a single‐stranded DNA or RNA molecule of defined sequence (the probe) can base‐pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs. Experimentally, the analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. Hybridization analysis is sensitive and permits detection of single‐copy genes in complex genomes. This unit presents a basic procedure for hybridization analysis with a radiolabeled DNA probe. Despite its lack of embellishment, the protocol gives acceptable results with Southern blots on nitrocellulose and nylon (uncharged and charged) membranes. An describes a similar method for probing DNA blots with a radiolabeled RNA probe. A for stripping blots to ready them for reprobing is also provided.

     
 
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Table of Contents

  • Basic Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled DNA Probe
  • Alternate Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled RNA Probe
  • Support Protocol 1: Removal of Probes from Hybridized Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled DNA Probe

  Materials
  • DNA to be used as probe
  • recipeAqueous prehybridization/hybridization (APH) solution, room temperature and 68°C
  • 2× SSC/0.1% (w/v) SDS
  • 0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
  • 0.1× SSC/0.1% (w/v) SDS, 68°C
  • 2× and 6× SSC (unit 10.6)
  • Hybridization oven (e.g., Hybridiser HB‐1, Techne) or 68°C water bath or incubator
  • Hybridization tube or sealable bag and heat sealer
  • Additional reagents and equipment for DNA labeling by nick translation or random oligonucleotide priming (unit 10.10), measuring the specific activity of and separating unincorporated nucleotides from labeled DNA (unit 10.9), and autoradiography ( appendix 3A)

Alternate Protocol 1: Hybridization Analysis of a DNA Blot with a Radiolabeled RNA Probe

  Additional Materials
  • Plasmid vector containing DNA to be transcribed into the RNA probe (Table 10.6.1)
    Table 0.6.1   Additional MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases

    Vector Size (bp) Markers a Promoters
    pBluescript 2950 amp, lacZ′ T3, T7
    pGEM series 2746‐3223 amp, lacZ′ SP6, T7
    pGEMEX‐1 4200 amp SP6, T3, T7
    pSELECT‐1 3422 tet, lacZ′ SP6, T7
    pSP18, 19, 64, 65 2999‐3010 amp SP6
    pSP70, 71, 72, 73 2417‐2464 amp SP6, T7
    pSPORT1 4109 amp, lacZ′ SP6, T7
    pT3/T7 series 2700, 2950 amp, lacZ′ T3, T7
    pWE15 8800 amp, neo T3, T7
    pWE16 8800 amp, dhfr T3, T7

     aAbbreviations: amp, ampicillin resistance; dhfr, dihydrofolate reductase; lacZ′, β‐galactosidase α‐peptide; neo, neomycin phosphotransferase (kanamycin resistance); tet, tetracycline resistance.
  • TE buffer, pH 8.0 ( appendix 2A)
  • recipeLabeling buffer
  • recipeNucleotide mix
  • 200 mM dithiothreitol (DTT), freshly prepared
  • 20 U/µl human placental ribonuclease inhibitor
  • [α‐32P]UTP: 20 mCi/ml (800 Ci/mmol) or 10 mCi/ml (400 Ci/mmol)
  • SP6 or T7 RNA polymerase
  • RNase‐free DNase I (unit 10.11)
  • 0.25 M EDTA, pH 8.0 ( appendix 2A)
  • recipeFormamide prehybridization/hybridization (FPH) solution
  • 2× SSC (unit 10.6) containing 25 µg/ml RNase A and 10 U/ml RNase T1
  • Additional reagents and equipment for purifying plasmid DNA (unit 10.6), phenol extraction and ethanol precipitation (unit 10.1), restriction digestion of DNA (unit 10.8), measuring the specific activity of and separating unincorporated nucleotides from labeled RNA (unit 10.9), and autoradiography ( appendix 3A)

Support Protocol 1: Removal of Probes from Hybridized Membranes

  Additional Materials
  • recipeMild stripping solution
  • recipeModerate stripping solution
  • 0.4 M NaOH
  • 0.1% (w/v) SDS, 100°C
CAUTION: Although the stripping solutions may not become highly radioactive, they should still be disposed of as radioactive waste.
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Figures

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Literature Cited

Literature Cited
   Amasino, R.M. 1986. Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal. Biochem. 152:304‐307.
   Britten, R.J. and Davidson, E.H. 1985. Hybridisation strategy. In Nucleic Acid Hybridisation: A Practical Approach (B.D. Hames and S.J. Higgins, ed.) pp. 3‐46. IRL Press at Oxford University Press, Oxford.
  Brown, T.A. (ed.) 1991. Molecular Biology Labfax. BIOS Scientific Publishers, Oxford.
   Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes as high concentrations of formamide. Nucl. Acids Res. 4:1539‐1552.
   Dyson, N.J. 1991. Immobilization of nucleic acids and hybridization analysis. In Essential Molecular Biology: A Practical Approach, Vol. 2 (T.A. Brown, ed.) pp. 111‐156. IRL Press at Oxford University Press, Oxford.
   Hutton, J.R. 1977. Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea. Nucl. Acids Res. 4:3537‐3555.
   Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. 1984. Improved technique utilizing non‐fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1:3.
   Little, P.F.R. and Jackson, I.J. 1987. Application of plasmids containing promoters specific for phage‐encoded RNA polymerases. In DNA Cloning: A Practical Approach, Vol. 3 (D.M. Glover, ed.) pp. 1‐18. IRL Press at Oxford University Press, Oxford.
   Meinkoth, J. and Wahl, G. 1984. Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138:267‐284.
   Mundy, C.R., Cunningham, M.W., and Read, C.A. 1991. Nucleic acid labelling and detection. In Essential Molecular Biology: A Practical Approach, Vol. 2 (T.A. Brown, ed.) pp. 57‐109. IRL Press at Oxford University Press, Oxford.
   Singh, L. and Jones, K.W. 1984. The use of heparin as a simple cost‐effective means of controlling background in nucleic acid hybridization procedures. Nucl. Acids Res. 12:5627‐5638.
   Wahl, G.M., Stern, M., and Stark, G.R. 1979. 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. 76:3683‐3687.
   Wetmur, J.G. and Davidson, N. 1986. Kinetics of renaturation of DNA. J. Mol. Biol. 31:349‐370.
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