Method of the Month — Southern Blotting
November 2021
Our third and final installment of the “gel-based” methods is Southern Blotting. This technique uses restriction enzymes, gel electrophoresis, and simple diffusion to confirm the identity of a DNA target strand. Check out our other articles on Western Blotting, Northern Blotting, and gel electrophoresis if you want to learn more about these foundational molecular biology techniques.
The detection of a target DNA sequence from a cell’s full genome is a powerful technique. It can show the presence of a gene encoding a potent toxin in a strain of bacteria or confirm the success of a cloning experiment in E. coli. It can even alert clinicians to harmful mutations in human cells. The secret? Paper towels. It may sound too simple to be true, but the Southern Blot technique relies on the capillary action of paper towel absorption to transfer a target strand of DNA to nitrocellulose paper with great effectiveness.
The first step is to break down DNA into more manageable pieces with restriction enzymes. A Southern Blot typically starts with a full cell genome, so the size of DNA strands must be reduced. Restriction enzymes cut DNA at a particular 4 to 6 base-pair sequence, such as ATCG. Every time the enzyme finds an ATCG, it cleaves that site and cuts the DNA strand in half. This is known as a restriction digest, and it can be carried out with one or more restriction enzymes to cut the DNA at different sequences.
Next, the digested DNA is loaded into a gel and electrophoresis performed to separate the sample into fragments of different lengths. Now, even if you know the length of the target strand, and that same length is identified, you still can’t be sure whether the sequence is the target strand or not. This is where the Southern Blot’s signature technique truly begins.
Much like a Northern Blot, the DNA strands can easily migrate through the gel, but since gels are not the best for further analysis, you have to get your samples onto a nitrocellulose membrane. A “gel sandwich” is constructed in the following manner:
The DNA in the gel is carried by buffer into the nitrocellulose, but while the buffer can easily diffuse upward into the paper towels, the DNA cannot and gets stuck in the nitrocellulose membrane. The upward diffusion may seem counterintuitive, but think of the last time you cleaned up a spill with paper towels. The liquid was able to climb up the paper towel against gravity by capillary action. Instead of applying a charge as in Northern Blotting, Southern Blots use nothing more than this wonderfully simple method of moving liquid upward against gravity.
Before further analysis, it is important to heat the system to denature the DNA into single strands, and then add a mixture of proteins, polysaccharides, and detergent to prevent non-specific binding to the DNA strands (“blocking”).
Once blocking is complete, a strand complementary to the target strand is washed over the membrane. The complementary strand is added to bind and “paint” the target strand with fluorescence or radioactivity. This confirms the identity of the strand and shows its size in base pairs due to the gel separation. The nitrocellulose membrane looks almost identical to that which is generated by a Northern Blot, but it contains DNA instead of RNA.
To review, the Southern Blot consists of four basic parts:
- Restriction digest
- Gel electrophoresis
- Transfer of DNA from the gel to membrane via simple diffusion
- Binding of marked complementary DNA to the target strand after heating and blocking.
These steps are all fairly simple molecular biology techniques, but their combination in the Southern Blot makes for a powerful tool that can be used by almost anyone with basic lab skills.
For more details on Southern Blotting, check out this source article.
