Abstract
The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the following steps: Size-fractionated DNA is first transferred from a gel matrix to a solid support under conditions that prevent self-annealing. The DNA molecules are then immobilized (covalently linked to the support) and processed for hybridization to a radiolabeled probe that has a nucleotide sequence complementing that of the target sequence to be detected. The blot is then washed extensively to remove unreacted probe molecules, and the hybrids formed between the probe and target sequences are revealed by autoradiography. The relative intensity of each autoradiographic signal will reflect the amount of hybridized material present. Quantification may be performed visually, by scanning laser densitometry, or, if the hybrids are sufficiently radioactive, by liquid scintillation counting.
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References
Meinkoth, J. and Wahl, G. (1988) Analytical strategies for the use of DNA probes. Anal. Biochem. 169, 1–25.
Southern, E. N. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. jI Mol. Bid. 98, 508–517.
Fourney, R. M., Dietrich, R. D., and Paterson, M. C. (1989) Rapid DNA extraction and sensitive alkaline blotting protocol: Application for detection of gene rearrangement and amplification for clinical molecular diagnosis. Disease Markers 7, 15–26.
Reed, K. C. and Mann, D. A. (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13, 7207–7221.
Feinberg, A. B. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, G13.
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© 1991 The Humana Press Inc., Clifton, NJ
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Fourney, R.M., Aubin, R., Dietrich, K.D., Paterson, M.C. (1991). Determination of Foreign Gene Copy Number in Stably Transfected Cell Lines by Southern Transfer Analysis. In: Murray, E.J. (eds) Gene Transfer and Expression Protocols. Methods in Molecular Biology, vol 7. Humana Press. https://doi.org/10.1385/0-89603-178-0:381
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DOI: https://doi.org/10.1385/0-89603-178-0:381
Publisher Name: Humana Press
Print ISBN: 978-0-89603-178-4
Online ISBN: 978-1-59259-494-8
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