Sunday, 11 April 2021

Southern blotting - Basic principle & Steps

 Blot refers to the membrane, on which biological molecules such as proteins and nucleic acids are absorbed or immobilised. The process of transferring these molecules from a gel to a membrane followed by their detection on the membrane is known as blotting

  When the macromolecule involved is DNA the technique is known as southern blotting. Southern is the last name of the scientist who first blotted DNA. He is Sir Edwin Mello southern.

  By analogy blotting involving RNA is known as northern blotting and for protein this technique is known as Western blotting. Both southern and northern blotting techniques are based on nucleic acid hybridization.

Southern Blotting

  Southern blotting has been used for the detection of a specific DNA fragment in a given mixture of DNA fragments or total cell.    

  Suppose we have isolated genomic DNA from bacteria. Now we want to find out whether gene or DNA sequence of interest is present in this bacterial genome or not. The researcher already knows the nucleotide composition of the target DNA sequence.

Step 1. DNA Digestion

  In the first step, the bacterial DNA is digested with a restriction enzyme. This step results in thousands of DNA fragments of various sizes. This is because the restriction enzyme cuts the chromosomal DNA at many different sites within the chromosome. 

  Again the researcher already knows that the target DNA sequence would be present within a specific restriction enzyme site. Therefore that restriction enzyme is used for the step of DNA digestion.

Southern blotting technique

Step 2. DNA Gel Electrophoresis

   The second step is the DNA gel electrophoresis. The separation of DNA fragments resulting from the first step is done by gel electrophoresis. The DNA fragments get separated according to their molecular weights.

   These separated DNA fragments are double-stranded but for hybridization step we require single-stranded DNA fragments. Therefore before moving to the next step these DNA fragments are denatured in the gel by exposure of Delta mild alkali(NaOH).

  The gel is soaked in a denaturing solution which contains sodium hydroxide. Sodium hydroxide denatures the DNA by disrupting hydrogen bonds between the two complementary strands.

Step 3. Blotting

   In the third step, bloating is done. This means the DNA fragments are transferred from the gel to a suitable membrane. This membrane can be nitrocellulose membrane or nylon membrane.

  Nowadays the most preferred membrane for such transfer of nucleic acids is nylon membrane. This is because it has a high tensile strength and better binding capacity for nucleic acids.

How this transfer is carried out ?

  There is the traditional way to transfer the DNA to the membrane. The basis of this transfer is the capillary action.  

System of southern blotting

  The tray is filled with the suitable transfer buffer. A support for gel on membrane is kept in this buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. These are placed such that they imbibe the buffer. They act as a wick

-  Next the gel containing DNA is placed on the top of it.
-  An nylon membrane of the same size as that of the gel is placed over it.
- Again a thick stack of blotting papers or absorbent paper is placed over the membrane.
- This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.

  From the tray now Rises through the gel taking with it the DNA molecules. Once the DNA molecules reach the nylon membrane they become adsorbed tightly to the membrane.

  The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. During this transfer the DNA fragments retain the same pattern of separation they had on the gel.  

   Another method to transfer DNA is by using the electric field. this method is similar to the Western blotting. The difference is that here we are transferring negatively charged DNA molecules from the gel to the membrane instead of proteins.

Step 4. Hybridization and Washing

    The next step is hybridization and washing. In this step the nylon membrane is now incubated with many copies of a nucleic acid probe. Probe is a labeled single-stranded DNA molecule. 

   Under appropriate conditions probe will bind to the target sequences pairing and form a double stranded DNA hybrid. So the probe hybridizes with the DNA fragment that is complementary to it. Unbound probes are removed by washing

Step 5. Detection

   In the fifth step detection of the bound probe is carried out. We find the location of the double stranded hybrid formed in the previous step. 

  The probe is detected by autoradiography, fluorescence or a color change depending on what label we have used in the probe.

Importance

  Southern blotting is useful for detecting major gene arrangments. This technique plays important role for example,
- In DNA fingerprinting,
- Identification of noval gene,
- Identification of structurally related genes in the species, etc.

Summary 

•  In southern blotting a mixture of DNA fragments is prepared by restriction digestion.
•  These DNA fragments are then separated by gel electrophoresis.
•  Next they are transferred and immobilised to a solid support such as nylon membrane.
•  Once immobilised the DNA fragments present on the membrane are now incubated with a specific probe.
•  The hybridization takes place between the probe and the DNA fragment that has complementary base pairs.
•  In the last step the location of the hybrid is detected by detection of the probe.

0 comments:

Post a Comment

I appreciate that you like our post and wants to comment but don't add any links between comments.