Southern Hybridization is the technique which was first given by the scientist E. M. Southern in 1975. It is a type of blotting method, which involves a transfer of the DNA from the solid agarose gel to the adsorbent medium like nitrocellulose or nylon filter paper.
Southern blotting is done after the process of DNA hybridization, where the target DNA is first cleaved by the restriction endonuclease. The target ss-DNA then complementary pairs with the radioactive label DNA probe to form a ds-DNA which can be visualized by the X-ray film.
Content: Southern Hybridization
- Definition of Southern Hybridization
- Principle of DNA Hybridization
- Transferring mechanism of Southern Hybridization
- The process of Southern Hybridization
Definition of Southern Hybridization
Southern hybridization also refers as Southern blotting. It can define as the method of isolating target DNA or desired gene of a sequence by labelling it with the complementary radioactive probe. The probe will bind with the target DNA which can visible on the X-ray film by the process refers as autoradiography.
Principle of DNA Hybridization
Southern hybridization is a method of isolating DNA of interest from the mixture of DNA molecules. This method is very much similar to the Restriction Fragment Length Polymorphism (RFLP).
Southern hybridization is based upon the principle of separating the target DNA by the method of probe hybridization and autoradiography that separates the target DNA.
Southern blotting is done after the separation of DNA fragments on the basis of length by electrophoresis. Then the southern hybridization is carried out to separate desired DNA by reacting it with a specific DNA probe which will make a complementary pair with the ss-DNA.
Transferring mechanism of Southern Hybridization
The replicas of DNA on the agarose gel is transferred to the nylon filter by the capillary mechanism. The capillary action moves the buffer solution upwards to the nitrocellulose or nylon filter, which will hit the DNA to be print on the filter. The paper towel and weight do not allow the migration of DNA from the nylon filter.
The gel is much fragile, whereas nylon filter is easy to handle and it is a quite sticky membrane where the DNA bands stick easily. The filter makes the further steps easier to perform like probe hybridization and autoradiography.
The process of Southern Hybridization
Southern blotting mainly involves the following seven steps:
First, cleave the DNA molecule into short fragments by the enzyme Restriction endonuclease.
Then separate the DNA of different size or length on the solid media like agarose gel, by the process of Gel Electrophoresis. The solid matrix provides the complex network for the migration of DNA fragments based on their size from a cathode to anode under the electric field. The larger DNA fragments will found close to the well whereas the smaller DNA fragments will move faster. Therefore, different bands of DNA will appear of varying length on the solid matrix.
After the band formation, treat the gel with the alkaline solution to break the ds-DNA into ss-DNA.
Southern blotting involves the transfer of the DNA bands from the agarose gel to the nitrocellulose filter paper. Blotting is a very crucial step which has to be performed with care which involves the following steps:
- First, take the alkaline buffer solution in the container.
- Then place a sponge over it.
- After that place the agarose gel consisting of different DNA bands.
- Then place nitrocellulose paper and over the top of filter add some paper towels and weight.
The transfer of DNA bands to the nitrocellulose filter is the process of forming a replica. The exact bands on agarose gel will now appear on the filter paper by the capillary action.
After blotting, bake the nitrocellulose filter membrane at 80 Degrees Celsius for up to 10 minutes.
Then hybridize the DNA bands on the nylon filter by adding radioactive probe in situ. The probe will bind with the desired DNA molecule by making it ds-DNA.
After the probe hybridization, wash the filter to remove the free probes. Then expose the nitrocellulose filter to the X-ray film. The X-ray will help us to visualize the hybridized or the desired DNA of interest on the nylon filter.
- One can detect the presence of DNA in the sample by Southern hybridization.
- By Southern blotting, the DNA or desired gene of interest can be isolated.
- One can also identify the mutation in the DNA sequence by the further study of target DNA.
- Southern hybridization is also used in the process of Restriction Fragment Length Polymorphism (RFLP).
Therefore we can conclude that the southern blotting is a method of separating nucleic acid (only DNA). Southern is a type of blotting technique or hybridization method where the target DNA complementary pairs with the radioactive DNA probe.
On the above, there is a flow chart of southern blotting which involves steps like restriction digest, gel electrophoresis, alkali treatment, blotting, baking, probe hybridization and autoradiography.