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Section 4 : Southern Blotting

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Southern Blotting, Page 1 of 2

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Abstract:

Southern blotting is the process of transferring and immobilizing DNA onto a membrane after restriction enzyme digestion and electrophoresis. The purpose is to subsequently probe the membrane with DNA to find complementary sequences. A complementary fragment of DNA is used to detect specific DNA sequences immobilized onto a membrane from a Southern blot. Two probes, DYZ1 and D2S44, are commonly used for identification of individuals. DYZ1 is a Y chromosome-specific probe, and D2S44 detects a restriction fragment length polymorphism (RFLP) on chromosome 2. The probe serves as a label or tag to identify specific regions of DNA out of the total 3 billion base pairs of DNA found in the human genome. Long, relatively intact pieces of DNA are required for restriction enzyme digestion for Southern blot analysis of human genomic DNA. Approximately 10- to 20-pg quantities are required to identify a single-copy region from the human genome. This section also presents an exercise on restriction fragment length polymorphism analysis of human DNA.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4

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Restriction Fragment Length Polymorphism
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Figures

Image of Figure 16.1
Figure 16.1

Overview of Southern blotting.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Image of Figure 16.2
Figure 16.2

DNA probe hybridizes with its complement.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Image of Figure 16.3
Figure 16.3

DNA probe does not hybridize to complementary DNA

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Figure 16.4

Diagram of assembly of the blot portion of a Southern blot.

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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References

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1. Ausubel, F. M.,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman,, J. A. Smith,, and K. Struhl (ed.). 1994. Current Protocols in Molecular Biology. Wiley Interscience, New York, N.Y.
2. Boehringer Mannheim. DIG DNA Labeling and Detection Kit. BoehringerMannheim Corp., Indianapolis, Ind.
3. Gentra Systems. Puregene DNA Isolation Kit. Gentra Systems, Inc., Minneapolis, Minn.
4. Kirby, L. T. 1992. DNA Fingerprinting: an Introduction. W. H. Freeman & Co., New York, N.Y.
5. Maniatis, T.,, E. F. Fritsch,, and J. Sambrook. 1982. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
6. Nakahori, Y. 1986. A human Y-chromosome with specific repeated DNA family (DYZ1) consists of a tandem array of pentanucleotides. Nucleic Acids Res. 14:75697573.
7. Nakamura, Y.,, M. Leppert,, P. O'Connell,, R. Wolff,, T. Holm,, M. Culver,, C. Martin,, E. Fujimoto,, M. Hoff,, E. Kumlin,, and R. White. 1987. Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235:16161622.
8. Odelberg, S. J.,, R. Plaetke,, J. R. Eldridge,, L. Ballard,, P. O’Connell,, Y. Nakamura,, M. Leppert,, J.-M. Lalouel,, and R. White. 1989. Characterization of eight VNTR loci by agarose gel electrophoresis. Genomics 5:915924.
9. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503517.

Tables

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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
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Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. Southern Blotting, p 141-156. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch4

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