Southern Blotting

Southern blotting was named after Edward M Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s.

Definition A technique for detecting specific DNA sequences following agar gel electrophoresis of a set of DNA restriction enzyme digestion fragments.

Principal Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome

Procedure of Southern Blotting

Digest the DNA Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. Run the digest The DNA fragments are then electrophoresed on an agarose gel separate them by size

Denature the DNA If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces. Fragments greater than 15 kb are hard to transfer to the blotting membrane alkaline transfer methods can also be used to denature the DNA, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA

Transfer the denatured DNA to the membrane A sheet of nitrocellulose membrane is placed on top of the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane

CONTI…….. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane

Expose The Membrane to Hybridization probe Membrane is Baked The membrane is then baked, i.e., exposed to high temperature (60 to 100 °C) (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently and covalently crosslink the DNA to the membrane. Expose The Membrane to Hybridization probe The membrane is then exposed to a hybridization probe, a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined Probe is labeled with radio active material

Visualization After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography Result Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe.

Tris-acetate buffer: Fermentas 50X TAE diluted to 1X concentration. Denaturation solution: 1.5 M NaCl, 0.5 M NaOH. Neutralization solution: 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA. 20X SSC solution (blotting buffer): 3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA. 100X Denhardt's solution: 2% (w/v) BSA (bovine serum albumin), 2% (w/v) Ficoll, 2% (w/v) PVP (polyvinylpyrrolidone). Hybridization solution: 5X SSC, 5X Denhardt's solution, 40% formamide, 0.5% SDS. 1 mg/ml sonicated herring sperm DNA solution. 10% (w/v) SDS solution.

What Southern blotting can tell us? Whether a particular gene is present and how many copies are present in the genome of an organism The degree of similarity between the chromosomal gene and the probe sequence

Whether re-arrangements have occurred during the cloning process Whether recognition sites for particular restriction endonucleases are present in the gene. By performing the digestion with different endonucleases, or with combinations of endonucleases, it is possible to obtain a restriction map of the gene i.e. an idea of the restriction enzyme sites in and around the gene- which will assist in attempts to clone the gene

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