Southern Blotting

1)    Southern hybridization DNA-DNA:

The southern blotting or the DNA blotting technique is being invented by the Edward M Southern of the Oxford University in 1970s and use specifically to identify specific sequence of DNA. The detected DNA could be a single gene or a large part of DNA like the genome of the virus. This techniques was later develop into the RNA and protein( northern and western blotting).

Goals of southern hybridization:

•       Immobilization of DNA onto the surface of permanent substrate, like on the nitrocellulose                    membrane.
•   Identification of the sequence of interest in DNA.

Pathway of southern blotting:

The blotting process or the transfer of the desire nucleotide sequences can be carry out by the standard process known as the gel electrophoreses. In this process the DNA is extracted from thesource, the isolated DNA is then cut with the restriction endonucleases into small piece at specific sites; these small pieces also have the gene of interest which we want to hybridize. Using gel electrophoresis technique the fragments of various lengths can be separated, commonly used gel is agars and acrylamide gel. 

 Southern blotting pathway

Sample is loaded in run in the gel; the gel is delicate structure, which may not be further used for analytical studies so the DNA fragments are immobilized on the nitrocellulose nylon membrane through the technique known as the southern blotting. In this process the arrangement of the, alkaline buffer at the bottom in the tray, sponge, agarose gel, nitrocellulose nylon membrane and a paper towel is placed at the top, some pressure is provide from the top of the setup, the DNA fragments move from bottom to the nylon membrane through the capillary action and became immobilize there. 

Capillary action shifts DNA from agarose gel to nitrocellulose paper

Now, the probes of specific known sequence complementary to desire strand labeled with detector is added to the nylon membrane, which have affinity for DNA, remember the nylon containing DNA is now placed in the plastic bag. The probe will bind only to the specific strand non-covalently during incubation period, it not bind to other strand present there which are not complement. The excess probe is washed off, the annealed probe remain on the nitrocellulose membrane. After the probe is washed off the paper is overlaid with X-ray film. The film and nitrocellulose membrane are kept in dark for a period of time, the radioactivity of the probe creates black bands on the X-ray film which is detected by an auto radiograph. The autoradiograph use X- ray film to detect radioactive material. The permanent record of the positions and the intensities of labeled band can be produce through this. So, the hybrid strands could be detected in this way. The reaction of antibody with protein is analyzed through western blot.

Annealing of probe with complementary DNA only

Application of southern blotting:

• Southern blotting is used for the detection of DNA in a given sample.
• The example of southern blotting is DNA finger printing.
• Paternity testing, victim identification and criminal identification is possible through this.
• Desire gene Isolation and identification.
• The mutation and gene rearrangement of gene in DNA sequence could be identify through this.
• The diseased caused by genetic defects could be diagnosed through this.
• Used in infectious agents identification.