Introduction
Scientists are often portrayed as serious, objective individuals who perform experiments and analyse data tirelessly. While this is not a completely inaccurate depiction, we must not forget that creativity is a key part of the process of experimental design and research, in addition to having a light-hearted reaction to when something does not go according to plan.
Perhaps this wit is the reason why in the late 20th century a phenomenal instance occurred where humor and inventiveness were at play in the field of molecular biology. I will explain how this resulted in the naming of four different analytical techniques known as Southern, northern, western and eastern blotting. Although, you probably will have noticed the irony in those titles already.
The original: Southern blotting technique
Edward Southern (born in 1938) is a British molecular biologist who has worked in the University of Edinburgh and Oxford University. Among his greatest accolades is the design of a DNA probing technique which carries his name: Southern blotting.
This consists on finding a particular sequence of DNA after gel electrophoresis by using a complementary fluorescent probe of DNA. While this may not seem very insightful, it is useful to identify which genes are present in a cell under different conditions (a healthy one versus a diseased one).
The method to conduct this is relatively simple, as you only have to digest the genome of your cell of interest via restriction enzymes, run a denaturing gel (so that the DNA sequences are made up of only one strand), transfer the DNA to a nitrocellulose membrane and then incubate this with your radioactive probe of interest. A diagram is shown below illustrating this process.
Once you image the nitrocellulose membrane, only those DNA fragments which were complementary to the probe that was added will be visible.
Gel electrophoresis
As an aside, I am going to explain in a bit more detail what a gel electrophoresis is and how it works. If you are already familiar with this procedure, you can skip to the next section.
The objective of a gel electrophoresis is to separate molecules according to size. As the name indicates, a sample is placed on a gel, which, when subjected to an electrical current induces the movement of the molecules in the sample. In the case of DNA where the phosphate backbone is negatively charged, it moves from the cathode (negative) to the anode (positive) of the tank in which the gel is placed.
The reason why the separation occurs on the basis of size is because the gel is a meshwork. Smaller molecules will be able to move more rapidly through the pores, while larger molecules will get tangled in the meshwork and take longer to traverse the gel. Below is a visual representation of gel electrophoresis.
Gel electrophoresis can also be used to separate proteins. If Sodium Dodecyl Sulfate (SDS) solution is applied to the proteins before pipetting them onto the gel, they will denature and move along the gel, like DNA.
To carry out Southern blotting, the DNA must be denatured so that the probe can attach to one strand of the gene by base pairing complementarity.
The witty joke: northern blotting technique
If you were waiting for the punchline of the joke, here it is. Shortly after the Southern technique was published in 1975, another group of scientists came up with an analogous analysis. Instead of looking for DNA sequences, the new method probed RNA sequences. Of course, they could not come up with a better name for it than northern blotting.
Northern blotting allows researchers to find which RNA fragments are present in cells at a given time. The difference with respect to Southern blotting is that RNA is dependent on gene expression. Therefore, if a cell contains more or less mRNA for a specific gene than a healthy cell, this may be caused by a disease, since there is more or less of that protein being synthesised.
The experimental method is almost identical, with the exception of the nucleotides being used (ribonucleotides instead of deoxyribonucleotides).
I recommend watching this video if you are interested in learning more about how the two techniques mentioned above are carried out.
A clever twist: western blotting technique
After the sensation created by those two techniques, another biomolecule was added to the increasing repertoire of gel probing experiments. In this case, it was proteins and the cardinal point assigned to them was the west.
In previous articles, I have discussed the structure and function of certain proteins, such as adrenaline receptors, ATP synthase and those involved in vesicular transport. However, I have not addressed how these are studied.
Western blotting, one among many ways to study proteins, relies on the same principles highlighted before, but makes use of fluorescent antibodies instead of complementary probes.
The technique involves a more complex series of steps, often requiring more than one antibody, as shown above, and can take a very long time to set up properly.
The objective is to analyse the quantity of protein being expressed in a given cell (as well as whether it is absent or present). Imagine that a cancerous cell has a damaged mechanism for producing one of the proteins needed for cell cycle regulation. A Southern blot would be positive as the gene is still in the DNA. A northern blot could also be positive if the gene were transcribed, but for some reason not properly processed before translation started. However, in a western blot, assuming you have successfully carried out the steps and included proper controls, you should see no bands for that protein in the hypothetical cancerous cell.
One last hurrah: eastern blotting technique
As you can see from the image in the introduction, there are many more techniques that have been named in accordance to this “blotting compass”, but I will finish off with eastern blotting, as the others are variations on these four.
In fact, eastern blotting is already quite similar to western blotting, only with a slight change of focus. Instead of searching for a protein and how much of it is expressed, this method aims to identify post translational modifications (PTM) of proteins, mainly glyocosylation patterns. PTM are additions or trimmings of proteins once they have been synthesised by ribosomes, which can include addition of lipids, carbohydrates, hydroxyl groups, methyl groups, acetyl groups, or even other proteins, such as ubiquinone. Each one of these modifications alters the function of the protein and therefore increases the diversity of what proteins can achieve, as well as provide regulatory mechanisms (for instance, ubiquitination signals a protein’s degradation).
Conclusion
If you have not managed to laugh out loud by this “molecular biology joke”, I hope you have at least learnt a few new methods for analysing various biomolecules.
The key take away is that all of these techniques make use of gel electrophoresis, transfer onto a nitrocellulose membrane and some kind of fluorescent or radioactive probe that binds to a specific fragment of interest from one of the biomolecules. Once this nitrocellulose membrane is imaged, we can trace which samples contain the molecule we were looking for, which in turn allows us to reach meaningful conclusions which can relate physiological conditions to biological function, via chemical principles.
Bibliography
- Biomedical and Biological Sciences, 2017. The Principle of Southern Blotting and Northern Blotting, Blotting Techniques, The Full Mechanism. Available at: https://www.youtube.com/watch?v=RvbzjYM_Ok0
- Loftus, S., n.d. Southern Blot. [online] Genome.gov. Available at: https://www.genome.gov/genetics-glossary/Southern-Blot
- Mahmood, T. and Yang, P., 2012. Western Blot: Technique, Theory, and Trouble Shooting. [online] NCBI. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
- Southern, E., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98(3), pp.503-517.
- Cover image credits: https://blog.universalmedicalinc.com/using-agarose-in-gel-electrophoresis/
Lilly Pubillones
Trinity College Hartford
Biochemistry
Sin respuestas todavía