How Does Southern Blotting Work

Southern blotting is a technique used for the detection of specific DNA fragment within a sample. The blotting technique involves the separation of DNA fragments based on size via gel electrophoresis, transferring the size-fractionated DNA sample onto a filter membrane, hybridizing the specific DNA fragments with a labeled, sequence-specific probe, and detecting the labeled bands.

In addition to the identification of specific DNA in a sample, the size and the abundance of a particular type of DNA can also be determined by Southern blotting. The process involved in Southern blotting is described in this article.

Key Areas Covered

1. What is Southern Blotting
     – Definition, Principle, Applications
2. How Does Southern Blotting Work
     – Process of Southern Blotting

Key Terms: DNA, Gel Electrophoresis, Hybridization Probe, Nylon Membrane, Restriction Digestion, Southern Blotting 

What is Southern Blotting

Southern blotting is a hybridization technique used in the identification of specific DNA in a sample. The technique was first developed by Edward M. Southern in 1975. This process identifies DNA sequences, size, and abundance of a particular DNA sequence within a sample.

A Southern blotting membrane is shown in figure 1.

Figure 1: Southern Blotting Membrane

Applications of Southern Blotting

Applications of Southern blotting are described below.

1. To detect a particular DNA fragment
2. To isolate a particular DNA fragment
3. To identify mutations and gene rearrangements
4. In RFLP assays (restriction fragment length polymorphism)
5. To identify genetic diseases
6. To identify infectious agents
7. Paternity testing
8. In DNA fingerprinting to identify criminals

How Does Southern Blotting Work

Southern blotting involves the separation of DNA fragments based on size via gel electrophoresis, transferring them into a membrane, hybridizing them with a labeled, sequence-specific probe, and detecting the labeled bands. The steps of Southern blotting are described below.

1. DNA digestion – DNA sample is digested by restriction enzymes to obtain DNA fragments, and these fragments are amplified by PCR.
2. Gel electrophoresis – DNA fragments are size-fractionated by gel electrophoresis.
3. Denaturation – The gel with fractionated DNA is soaked in NaOH or HCl to denature double-stranded DNA.
4. Blotting – DNA fragments in the gel are transferred into a positively-charged nylon membrane by a process called blotting.
5. Baking and blocking – The blotting paper with DNA fragments is baked to fix DNA on the membrane. Then, the unoccupied binding sites are saturated by treating with Bovine serum albumin (BSA) or casein.
6. Hybridization with labeled probes – DNA bound membrane is treated with a labeled probe that contains complementary nucleotide sequence to the interested DNA fragment. Hybridization probes are generally 100-500 bp long, and they are labeled with radioactive nucleotides.
7. Visualization by autoradiogram – The probe bound membrane is visualized under autoradiogram to obtain the patterns of the interested DNA fragment.

Figure 2: Southern Blotting

The whole process of Southern blotting is shown in figure 2.

Conclusion

Southern blotting is a hybridization technique used in the identification of specific DNA sequences from a sample. In this process, the DNA sample is fractionated by restriction enzymes, and the mixture is run on a gel. Then, the banding pattern in the gel is transferred into a nylon membrane and hybridized with a labeled probe, which allows the detection of the interested fragment.

Reference:

1. “Southern Blotting.” Thermo Fisher Scientific, Available here.

Image Courtesy:

1. “Southern blot membrane” By Bojan Žunar – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Figure 17 01 05” By CNX OpenStax –  (CC BY 4.0) via Commons Wikimedia