Biochemical Techniques and Instrumentation

5.1.3. Blotting Techniques

Steps in Southern Blotting

There are eight major steps in southern blotting:

1.Restriction digestion: The genomic DNA is first digested with restriction endonucleases, which cut high-molecular-weight DNA strands into smaller fragments.

2.Agarose gel electrophoresis: The DNA fragments are then separated based on their size by an agarose gel electrophoresis. The gel is then washed with buffer to remove the broken or fragmented residues of agarose and contaminants.

3.Acid treatment: If DNA fragments are large in size (>15 kb), it takes longer time to transfer to the membrane compared to shorter DNA fragments. This depurination step with an acid (0.25M HCl for 15 min) takes the purines out and breaks the DNA into smaller fragments to allow efficient transfer to the membrane. The acid is then neutralized after this step.

4.Alkali treatment: After acid treatment, the gel is placed in 0.25 M NaOH alkali solution to denature the double-stranded DNA into single stranded DNA strands. This improves the binding of the negatively charged DNA to a positively charged membrane during hybridization. It also destroys any residual RNA that are present in the DNA. The gel is then washed with buffer to remove the traces of NaOH.

5.Blotting: The denatured DNA in the gel is then placed on the filter paper with wigs dipped in a reservoir containing transfer buffer, Sodium Saline Citrate (SSC). Nitrocellulose or nylon membrane is used for transfer of DNA from the gel. Nitrocellulose has a binding capacity of 100µg/cm, while nylon has a binding capacity of 500 µg/cm. Nylon is less fragile and binds more DNA than nitrocellulose membrane. The membrane is placed on the gel, above which a stack of blotting papers soaked in transfer buffer is placed and gently pressed using glass rod to remove the air that is trapped between the gel and membrane. Then a stack of un-soaked blotting papers and a weight of 500 g is placed above them. The transfer of DNA from gel to the membrane can take place by three mechanisms.

1. Capillary system - The transfer buffer is transferred from a region of high water potential to a region of low water potential by capillary action. In this process, the DNA from the gel is transferred onto the membrane and  immobilized due to the ion exchange interactions between the negative charge of the DNA and the positive charge of the membrane. This set up is allowed to stand for 12-18 h and the membrane is taken out carefully.
2. Vacuum blotting system: In vacuum blot apparatus, ess, the vacuum sucks the transfer buffer through the membrane and the gel in a faster speed within an hour.
3. Electro transfer system: In this system, an electrical force is used to transfer the DNA from the gel onto the membrane.

6.Fixation: There are two widely used methods.

1. UV­ cross linking method: The membrane is exposed to UV rays particularly in case of nylon membrane, so that a covalent bond is formed between negatively charged phosphate and thymine residues present in the DNA and the positively charged amino groups on the surface of the nylon membrane.
2. Vacuum oven method: In this method, the nitrocellulose or nylon membrane is baked at 80°C in a vacuum oven or regular oven for 2 h. The use of vacuum oven is a preferrable due to the flammable nature of nitrocellulose membrane.

7.Hybridization: Hybridization is the technique in which the nucleic acid immobilized on the membrane is challenged with a known probe. If the probes are made of DNA, they are called as DNA probes. Probes contain single DNA fragment with a specific sequence that are labelled by incorporating radioactive ³² P labeled ATP or tagged with a fluorescent or bioluminescent or chromogenic dye. After the blotting technique is completed, the membrane is exposed to a DNA hybridization probe. If the sequence of DNA probe is complementary to nucleotide sequence on the membrane, it forms hydrogen bond and gets converted into hybrid DNA. This is called base pairing or hybridization. After the hybridization, the membrane is washed using SSC transfer buffer to remove the unbound probes, while bound probes remain attached.

8. Detection: Under optimal conditions, 0.1 pg of the DNA can be detected by probing. The regions of hybridization are detected by different methods.

a.Autoradiography method: If the probe is radiolabelled ³²P or fluorescent labelled, the pattern of hybridization is visualized on X-ray film

b.Biotin streptavidin method: If the probe is labelled by a non-radioactive chromogenic dye, the pattern of hybridization is visualized by development of color on the membrane and measurement by colorimeter.

c. Bioluminescence method: Bioluminescent visualization uses luminesence.