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SOUTHERN BLOT

JONATHAN LOCKE LABORATORY METHODS IN MOLECULAR BIOLOGY BIOL-546. SOUTHERN BLOT. SOUTHERN BLOT. 1. 1953 - DNA structure 1966 - Agarose gel electrophoresis 1970 - Restriction enzymes 1975 - Southern Blot. Sir Edwin Mellor Southern.

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SOUTHERN BLOT

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  1. JONATHAN LOCKE LABORATORY METHODS IN MOLECULAR BIOLOGYBIOL-546 SOUTHERNBLOT

  2. SOUTHERN BLOT 1 1953 - DNA structure 1966 - Agarose gel electrophoresis 1970 - Restriction enzymes 1975 - Southern Blot Sir Edwin Mellor Southern Detection of specific sequences among DNA fragments separated by gel electrophoresis, E. M. Southern, Journal of Molecular Biology, Nov 1975 This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. Is a gene or fragment in my DNA? Why do this? Not able to routinely do PCR or sequencing in 1975. • Google Image Search

  3. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 1. Cleave DNA with restriction enzymes 1 • Need to find a restriction enzyme that will cut in the right places • Typically we cut out a gene, but any fragment can be cut out • Google Image Search

  4. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 2. Gel electrophoresis 1 Agarose gel electrophoresis 2 DNA fragments cut by restriction enzymes • Google Image Search • Ibid.

  5. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 3. Transfer DNA in gel by blotting onto nitrocellulose filter paper 1 Highly flammableToday - nylon membrane Capillary action 2 Bake blot at 80C for 2 hours • Google Image Search • Ibid

  6. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 4. Hybridize with labelled DNA complement (“probe”) Boil probe first to denature 1 Wash blot after hybridization • Google Image Search

  7. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS SIDEBAR: Where does the probe come from? Single-stranded complementary DNA is needed to hybridize with DNA on the filter 1 Originally, Southern used DNA-RNA hybridization of radioactive mRNA transcripts Today, we can easily sequence the gene and then either use universal probe libraries or create a custom probe • Google Image Search

  8. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS SIDEBAR: How does hybridization work in general? 1 • Heat denatures the DNA making it linear • Cooling allows hybrids to form • But also duplexes of the original samples In Southern blotting, the target DNA is not in aqueous solution. It is permanently denatured and fixed to the filter paper, so we only get hybrid duplexes. • Google Image Search

  9. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 1 • Google Image Search

  10. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 5. Visualization 1 2 3 • Then: • X-Ray • Today: • Fluorescent • Chromogenic - visible dyes • Lucinogenic - ? • Google Image Search • Ibid. • Ibid.

  11. SOUTHERN BLOT PROCEDURE DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 1 • Google Image Search

  12. SOUTHERN BLOT COUSINS 1975 - Southern blot - DNA 1977 - Northern blot - RNA 1980 - Western blot - Proteins (antibodies - “immunoblot”) 1980 - Southwestern blot - DNA binding proteins 1982 - Eastern blot - Post-translational modifications (PTMs)

  13. SOUTHERN BLOT USES Restriction Fragment Length Polymorphism (RFLP) - Disease Alleles 1 3 1 2 1 3 2 • Google Image Search

  14. SOUTHERN BLOT USES RFLP - Pedigrees 1 (recessive allele) a (dominant allele) A Heterozygous Homozygous dominant Homozygous recessive • Google Image Search

  15. SOUTHERN BLOT USES RFLP - DNA Fingerprinting, Forensics 1 VNTR - Variable Number Tandem Repeat • Repeated sequence • Length tends to vary among individuals • Examining many VNTR sites (using many lanes) produces a fairly unique fingerprint • Google Image Search

  16. SOUTHERN BLOT USES Zoo Blots and Garden Blots The Tre2 (USP6) Oncogene Is a Hominoid-Specific Gene 1 Can compare one gene or many Can compare any source Plants Organ tissues Can do zoo blots with related techniques like Northern blots and Western blots • Charles A. Paulding, The Tre2 (USP6) Oncogene Is a Hominoid-Specific Gene, PNAS, Mar 2003

  17. SOUTHERN BLOT Why do this? Not able to routinely do PCR or sequencing in 1975. Southern blotting is used less and less due to modern sequencing. Questions?

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