Southern Blotting: Principle
A Southern Blotting is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA. A hybridization probe is a short (100-500bp), single stranded DNA. The probes are labeled with a marker so that they can be detected after hybridization.
The method is named after the British biologist Edwin Southern, who first published it in 1975. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern’s name. As the label is eponymous, Southern is capitalized, as is conventional of proper nouns. The names for other blotting methods may follow this convention, by analogy.
Southern Blotting: Procedure/ Steps
- Restriction digest: by RE enzyme and amplification by PCR
- Gel electrophoresis: SDS gel electrophoresis
- Denaturation: Treating with HCl and NaOH
- Blotting
- Baking and Blocking with casein in BSA
- Hybridization using labelled probes
- Visualization by autoradiogram
Step I: Restriction digest
- Restriction endonucleases are used to cut high-molecular[1]weight DNA strands into smaller fragments.
Step II: Gel electrophoresis
- The DNA fragments are then electrophoresed on an agarose gel to separate them by size.
Step III: Denaturation
- 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. This depurinates the DNA fragments, breaking the DNA into smaller pieces, thereby allowing more efficient transfer from the gel to membrane.
- If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later hybridization to the probe, and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results.
Step IV: Blotting
- A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. 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 onto 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.
Step V: Baking and blocking
- The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.
Step VI: Hybridization with labelled probes
- 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.
Step VII: Visualization by Autoradiogram
- After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
- 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.
Application of Southern blotting:
- Southern blotting transfer may be used for homology-based cloning on the basis of amino acid sequence of the protein product of the target gene.
- Oligonucleotides are designed so that they are similar to the target sequence.
- The oligonucleotides are chemically synthesized, radiolabeled, and used to screen a DNA library, or other collections of cloned DNA fragments.
- Sequences that hybridize with the hybridization probe are further analysed, for example, to obtain the full length sequence of the targeted gene.
- Southern blotting can also be used to identify methylated sites in particular genes.
