Southern Blotting

Southern blotting Southern blotting was invented by Edwin Southern in 1975 , a biologist of Edinburgh University, UK. Southern blotting is a technique in which the DNA molecules are transferred from an agarose gel onto a membrane and used to locate a specific gene within an entire genome.

Steps in blotting Restriction digestion Agarose gel electrophoresis Washing Acid treatment Alkali treatment Blotting Hybridization Detection

1. Restriction digestion Genomic DNA is restriction digested with restriction endonucleases that cut high molecular weight DNA strands into smaller fragments. 2. Agarose gel electrophoresis DNA fragments are electrophoresed on an agarose gel to separate them by size. 3. Washing Gel is first washed with sodium saline citrate buffer to remove the broken or fragmented residues of agarose formed during banding and accumulated contaminants.

4. Acid treatment If DNA fragments are large in size (>15 kb), they require a longer time to transfer from the gel to membrane. Depurination with an acid (0.25M HCl) for 15 min takes the purines out, breaking the DNA into smaller fragments. 5. Alkali treatment Gel is placed in an alkali solution (0.25 M NaOH) to denature the double - stranded DNA into single DNA strands, so that it improves the binding of the negatively charged DNA to a positively charged membrane. It is useful for later hybridization to the probe and also destroys any residual RNA present in the DNA. 6. Washing Gel is extensively washed with SSC buffer to remove the traces of NaOH.

7. Blotting DNA in the gel is placed on the filter paper with wigs dipped in a reservoir containing transfer buffer. Nitrocellulose or Nylon membrane can be used for transfer. Nitrocellulose has a binding capacity of 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Membrane is placed on the gel. A stack of blotting papers soaked in transfer buffer is placed above it and gently pressed using glass plate or rod to remove the air that is trapped between the gel and membrane. A stack of un-soaked blotting papers is then placed above them and a glass plate with a weight of 500 g is placed above it.

Transfer mechanism 1. Capillary method Buffer moves from reservoir to blotting paper by capillary action. During this process, the DNA from the gel is transferred onto the membrane within 12-18 h and immobilized there due to the ion exchange interactions between negative charge of the DNA and positive charge of the membrane. 2. Vacuum blot apparatus A vacuum sucks the transfer buffer through the membrane within 1 h. 3. Electro transfer system An electrical force is used to transfer the DNA from the gel onto the membrane.

Fixation methods Membrane is taken out carefully and has to be fixed more strongly, as the binding of the DNA to the membrane is weak. 1. UV­ cross linking method Membrane is exposed to ultraviolet rays, so that a covalent bond is formed between phosphate and thymine residues present in the DNA and the positively charged amino groups on the surface of nylon membrane. 2. Vacuum oven method Nitrocellulose or nylon membrane is baked at 80°C in a vacuum oven or regular oven for 2 h. Use of vacuum oven is a preferrable due to the flammable nature of nitrocellulose membrane.

8. Hybridization A technique by which the nucleic acid immobilized on the membrane is challenged with a known probe. It is used to confirm the presence or absence of the specific DNA sequence. Probes DNA hybridization probe is made of a single DNA fragment with a specific DNA sequence. DNA is labeled by incorporating radioactive 32P labeled ATP or tagged with a fluorescent or bioluminescent or chromogenic dye. Labeled DNA contains sequences complementary to DNA present on the membrane. If the sequence of DNA probe is complementary, it forms hydrogen bonds or converts into hybrid DNA. This is known as base pairing or hybridization.

9. Washing After hybridization, the membrane is washed using SSC buffer to remove the unbounded probes, while bound probes remain attached 10. Detection Detection limit is 0.1 pg of the DNA. 1. Autoradiography method - If the probe is radiolabeled 32P or fluorescent labeled, the pattern of hybridization is visualized on X-ray film 2. Biotin streptavidin method - If the probe is labeled by a non-radioactive chromogenic dye, the pattern of hybridization is visualized by development of color on the membrane and measurement by colorimeter. 3. Bioluminescence method – If the probe is tagged with bioluminescent, the pattern is visualized by measuring luminesence using luminometer.

Applications Determine the number of sequences (e.g., gene copies) in a genome. Find out specific DNA sequence present in different animals. eg. Presence of insulin gene in sea anemone. Detect the Restriction Fragment Length Polymorphism (RFLP). Polymorphism refers to DNA sequence variation between individuals of a species. If the sequence variation occurs at the restriction sites, it could result in RFLP.  Detect the Variable Number of Tandem Repeat Polymorphism (VNTR). VNTR result due to unequal crossover. It is the molecular basis of DNA fingerprinting.