Abstract
This chapter describes the detection of specific DNA sequences by hybridization to a labeled probe of complementary sequence. This method is suitable for the detection of a wide range of DNA concentrations down to single-copy genes within mammalian genomic DNA (little more than 1 pg of hybridizing DNA in a total of 10 µg). In principle, hybridization consists of the annealing of a single-stranded, labeled nucleic acid probe to denatured DNA fixed to the filter. In practice, this method is designed to provide a balance between maximizing the specific signal and minimizing the nonspecific background.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ardeshir, F., Giulotto, E., Zieg, J., Brison, O., Liao, W. S. L., and Stark, G. R. (1983) Structure of amplified DNA in different Syrian hamster cell lines resistant to Mphos-phonacetyl)-L-aspartate. Mol. Cell Biol. 3, 2076–2088.
Litt, M. and White, R. L. (1985) A highly polymorphic locus in human DNA revealed by cosmid-derived probes. Proc. Natl. Acad. Sci. USA 82, 6206–6210.
Djabali, M., Nguyen, C., Roux, D., Demengeot, J., Yang, H. M., and Jordan, B. R. (1990) A simple method for the direct use of total cosmid clones as hybridization probes. Nucleic Acids Res. 18, 6166.
Sealey, P. G., Whittaker, P. A., and Southern, E. M. (1985) Removal of repeated sequences from hybridisation probes. Nucleic Acids Res. 13, 1905–1922.
Denhardt, D. T. (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641–646.
Wahl, G. M., Stern, M., and Stark, G. R. (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridisation by dextran sulphate. Proc. Natl. Acad. Sci. USA 76, 3683–3687.
Feinberg, A. P. and Vogelstein, B. (1993) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.
Laskey, R. A. and Mills, A. D. (1975) Quantitative film detection of 3H and 14C in poly-acrylamide gels by fluorography. Eur. J. Biochem. 56, 335–341.
Laskey, R. A. and Mills, A. D. (1977) Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitive film. FEBS Lett. 82, 314–316.
Blonden, L. A. J., den Dunnen, J. T., van Paassen, H. M. B., Wapenaar, M. C., Grootscholten, P. M., Ginjaar, H. B., et al. (1989) High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization. Nucleic Acids Res. 17, 5611–5621.
Davis, R. and Meuth, M. (1994) Molecular characterization of multilocus deletions at a diploid locus in CHO cells: association with an intracisternal-A particle gene. Somat. Cell Mol Genet. 20, 287–300.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Kelsell, R. (2000). Hybridization and Competition Hybridization of Southern Blots. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:163
Download citation
DOI: https://doi.org/10.1385/1-59259-038-1:163
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
eBook Packages: Springer Book Archive