Hybridization Analysis of DNA Blots

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.10
DOI:  10.1002/0471142727.mb0210s21
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”) that has been immobilized on a nitrocellulose and nylon (uncharged and charged) membrane support. The approach taken in this unit is to present as the basic protocol an unsophisticated procedure for hybridization analysis with a radiolabeled DNA probe. The alternate protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A support protocol for stripping blots for reprobing is also provided. The commentary describes modifications, including changes to prehybridization, hybridization, and wash solution formulations, and alterations to incubation times and conditions, the latter including a discussion of the wash conditions compatible with different degrees of stringency.

     
 
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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
  • 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
  • Aqueous 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 ( appendix 22)
  • 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 3.5), measuring the specific activity of labeled DNA and separating unincorporated nucleotides from labeled DNA (unit 3.4), and autoradiography ( appendix 3A)

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

  Additional Materials
  • TE buffer, pH 8.0 ( appendix 22)
  • 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 (unit 3.8)
  • RNase‐free DNase I (unit 3.12)
  • 0.25 M EDTA, pH 8.0 ( appendix 22)
  • recipeFormamide prehybridization/hybridization (FPH) solution
  • 2× SSC ( appendix 22) containing 25 µg/ml RNase A + 10 U/ml RNase T1 (unit 3.13)
  • Additional reagents and equipment for cloning and purifying plasmid DNA (Chapter 1), phenol extraction and ethanol precipitation (unit 2.1), restriction digestion of DNA (unit 3.1), measuring the specific activity of and separating unincorporated nucleotides from labeled RNA (unit 3.4), 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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Literature Cited

Literature Cited
   Amasino, R.M. 1986. Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal. Biochem. 152:304‐307.
   Anderson, M.L.M. and Young, B.D. 1985. Quantitative filter hybridisation. In Nucleic Acid Hybridisation: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 73‐111. IRL Press at Oxford University Press, Oxford.
   Bonner, T.I., Brenner, D.J., Neufield, B.R., and Britten, R.J. 1973. Reduction in the rate of DNA reassociation by sequence divergence. J. Mol. Biol. 81:123‐135.
   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.
   Denhardt, D.T. 1966. A membrane‐filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641‐646.
   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.
   Hyman, R.W., Brunovskis, I., and Summers, W.C. 1973. DNA base sequence homology between coliphages T7 and ϕ11 and between T3 and ϕ11 as determined by heteroduplex mapping in the electron microscope. J. Mol. Biol. 77:189‐196.
   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
   Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979. Determination of nucleic acid sequence homologies and relative concentrations by a dot blot hybridization procedure. Nucl. Acids Res. 7:1541‐1552.
   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.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   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.
Key Reference
   Dyson, N.J. 1991. See above.
  Provides a detailed account of factors influencing hybridization.
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