Southern Blotting Principle, Procedure and Application

Southern blotting

• The technique was developed by E.M. Southern in 1975.
• The Southern blot is used to detect the presence of a particular DNA fragment in a sample.
• The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

Principle
The key to this method is hybridization.
Hybridization: It is the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA.
There are 2 important features of hybridization:

• The reactions are specific-the probes will only bind to targets with a complementary sequence.
• The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Summary of procedure (Detailed Procedure)

1. Extract and purify DNA from cells
2. DNA is restricted with enzymes
3. Separated by electrophoresis
4. Denature DNA
5. Transfer to nitrocellulose paper
6. Add labeled probe for hybridization to take place
7. Wash off unbound probe
8. Autoradiograph

Application:

• To identify specific DNA in a DNA sample
• To Isolate desired DNA for construction of rDNA
• Identify mutations, deletions, and gene rearrangements
• Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
• In RFLP
• Used in phylogenetic analysis
• Diagnosis of HIV-1 and infectious disease
• In DNA fingerprinting:
  • Paternity and Maternity Testing
  • Criminal Identification and Forensics
  • Personal Identification