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.
Steps in southern blotting process include:
- DNA extraction.
- Restriction Digestion or DNA Fragmentation.
- Gel Electrophoresis
- Denaturation and Depurination
- Baking and blocking
DNA Fragmentation or Restriction Digestion
The isolated DNA is fragmented by using suitable restriction enzyme which cuts DNA at specific site generating fragments. Restriction enzymes are used to cut high-molecular-weight DNA strands into smaller fragments. The number of fragments of DNA obtained by restriction digest is amplified by PCR.
The DNA fragments (of unequal length) are then electrophoresed on an agarose gel/ Sodium Dodecyl Sulphate gel to separate them.
Denaturation and Depurination
Agarose gel/ Sodium Dodecyl Sulphate gel after electrophoresis is soaked in alkali (NaOH) or acid (HCl) to denature double stranded DNA fragments. Dilute HCl partially depurinate DNA which promotes higher efficiency transfer by breaking down fragments into smaller pieces. NaOH causes double stranded to become single-stranded (denaturation) making them suitable for hybridization. It is then neutralized with NaCl to prevent re-hybridization before adding probe.
The denatured fragments are then transferred to nylon or nitrocellulose filter membrane by the process of blotting in blotting stack.
Baking and blocking
The nitrocellulose membrane is removed from blotting stack and baked on autoclave to fix DNA of interest bound onto it. The membrane can also be baked in a vacuum or regular oven at 80°C for 2-3 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane.
The membrane is then treated with casein or Bovine serum albumin (BSA) which saturates all the binding site of membrane.
The DNA bound to membrane is then treated with hybridization probe which contains complementary sequences to the gene of interest. Probe is labeled with radioactive molecule, fluorescent or chromogenic dye so that it can be detected.
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. In some cases, the hybridization probe may be made from RNA rather than DNA
The conditions are so chosen that probe hybridizes with complementary DNA on the membrane to the greatest extent with a low nonspecific binding on membrane.
The membrane is thoroughly washed after hybridization with a buffer to remove unbound probes and probe that is bound non-specifically so that only labeled probe bound to target sequence remains in it.
The hybridized segments are detected autoradiographically by placing nitrocellulose membrane in contact with a photographic film. The images show pattern/bands of hybridized DNA molecules.
The pattern of hybridization is visualized on X-ray film by autoradiography in case of a radioactive or fluorescent probe or by development of color on membrane if a chromogenic detection method is used.