Southern blotting is an example of RFLP (restriction fragment length polymorphism). It was developed by Edward M. Southern (1975). Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labeled probe hybridization.
Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, are detected using specific DNA probe that is complementary to the desired DNA.
A hybridization probe is a short (100-500 bp), single stranded DNA. The probes are labeled with a marker so that they can be detected after hybridization.
- Restriction digestion by restriction endonucleases or restriction enzymes and amplification by PCR.
- Gel electrophoresis: SDS agarose gel electrophoresis.
- Denaturation: Treating with HCl and NaOH.
- Baking and Blocking with casein in BSA (Bovine Serum Albumin).
- Hybridization using labeled probes.
- Visualization by autoradiogram.
Step I: Restriction digest
- The DNA is fragmented by using suitable restriction enzyme. RE cuts the DNA at specific site generating fragments.
- The number of fragments of DNA obtained by restriction digest is amplified by PCR.
Step II: Gel electrophoresis
- The desired DNA fragments is separated by gel electrophoresis.
Step III: Denaturation
- The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature the double stranded DNA fragments.
- DNA strands get separated.
Step IV: Blotting
- The separated strands of DNA is then transferred to positively charged membrane nylon membrane (Nitrocellulose paper) by the process of blotting.
Step V: Baking and blocking
- After the DNA of interest bound on the membrane, it is baked on autoclave to fix in the membrane.
- The membrane is then treated with casein or Bovine serum albumin (BSA) which saturates all the binding site of membrane.
Step VI: Hybridization with labeled probes
- The DNA bound to membrane is then treated with labeled probe.
- The labeled probe contains the complementary sequences to the gene of interest.
- The probe bind with complementary DNA on the membrane since all other non-specific binding site on the membrane has been blocked by BSA or casein.
Step VII: Visualization by Autoradiogram
- The membrane bound DNA labeled with probe can be visualized under autoradiogram which give pattern of bands.
Applications of Southern blotting:
- Southern blotting technique is used to detect DNA in given sample.
- DNA finger printing is an example of southern blotting.
- Used for paternity testing, criminal identification, victim identification.
- To isolate and identify desire gene of interest.
- Used in restriction fragment length polymorphism.
- To identify mutation or gene rearrangement in the sequence of DNA.
- Used in diagnosis of disease caused by genetic defects.
- Used to identify infectious agents.