Welcome back! This is the second article in a Sidebar series where Manolya, Jamie, and I are offering background on common laboratory experiments. Last time, Manolya went over how the number of viruses in a sample can be estimated using a plaque assay. Today, I will be walking you through one of the most fundamental procedures used by biologists every day, called PCR.
If you have been reading news articles, scientific papers, or other Demystified posts discussing the latest inquiries on SARS-CoV-2, you probably have come across the term “PCR” more than once. Unfortunately, if you Google “PCR”, the top results that come up are often heavy on the jargon, and assume a lot of background knowledge in biology and chemistry.
To give a TL;DR summary:
(1) PCR stands for “Polymerase Chain Reaction”.
(2) PCR is a method of making lots of copies of a DNA molecule.
That’s it! It’s quite simple. But this explanation may leave you with a lot of questions. What’s so important about DNA anyway? How are the DNA copies made? Why do we even want these DNA copies? The idea behind this article is to try and answer these vital questions regarding PCR, without getting too deep into the biochemical details.
As a quick overview of what we will cover today:
(1) Thinking of DNA, RNA, and proteins as strings of characters, not molecules.
(2) Information flow from DNA to RNA to proteins.
(3) The machinery that cells use to make copies of their own DNA.
(4) How scientists borrow this cellular machinery in order to copy DNA of their choice.
In the near future, you can expect a Sidebar article from me that will describe two extra flavours of PCR, called RT-PCR and qPCR, which together are used for SARS-CoV-2 diagnostic testing. For now, let’s proceed with our crash course on DNA, RNA, proteins, and basic PCR!
To start: how to think about DNA, RNA, and proteins.
Lasya elegantly described the biochemistry of DNA, RNA, and proteins in her Sidebar, “On Mutation”. I’m going to reiterate some of what she talked about here, although I will be using a slightly different analogy that takes out the notion of molecules.
Let’s just think of DNA, RNA, and proteins as long strings of characters.
Although DNA, RNA, and proteins are all character strings, they each have defining characteristics.
- DNA has only 4 allowed characters, “A”, “C”, “G”, and “T”. DNA is usually double-stranded, meaning that the whole object actually consists of two character strands that have been paired up. “A” tends to pair with “T” on opposite strands, and “C” tends to pair with “G”. We call these characters complementary.
- RNA also has only 4 allowed characters, “A”, “C”, “G”, and “U”. For our purposes, we can think of RNA as being single-stranded, as I have shown in the figure above, although this is not always the case.
- Proteins look totally different, as they have 20 allowed characters.
What are the functions of DNA, RNA and proteins?
Now we have an idea of DNA, RNA, and proteins as different classes of character strings. But what’s the purpose of these character strings anyway?
For DNA and RNA, their main responsibility is to store information on how to build proteins.
DNA can be thought of as a complete manual, containing the instructions to construct all the different proteins in your body. Information from the DNA is transcribed into the RNA. You can think of the RNA as a particular chapter in the manual.
Since there are many chapters in the manual, multiple different RNAs can be made from one DNA. The information in the RNAs is translated in order to build various proteins.
Proteins can have many different functions. For example, hemoglobin is a protein that carries oxygen to your tissues. The tubulin protein is a critical structural element in all you body cells. The insulin protein is used as a chemical message to regulate your metabolism. The possibilities for protein function seem to be endless!
How is DNA copied?
At this point, we have a good grasp on DNA, RNA, and proteins as character strings. We also understand the flow of information from DNA to RNA to protein. But wait! We forgot about something along the way. How are these character strings even made?
First, let’s consider DNA. Every cell in your body has a DNA instruction manual. When a parent cell divides, it will make a copy of its DNA so that each new daughter cell receives a complete manual. This process is called DNA replication.
There are many protein machines that are involved in the copying of a DNA template. However, I will be focusing on just three:
(1) Helicase. Helicase will “unzip” the double-stranded DNA.
(2) Primase. Primase will read the characters in each DNA strand, and write a short sequence of characters that are complementary* to the template. These newly made regions are called primers.
* If you recall from earlier, “A” will pair with “T” and “G” will pair with “C”. Each of these pairs are complementary characters.
(3) DNA polymerase. DNA polymerase is a lot more efficient than primase at making new DNA. It will take over the complementary writing process and extend off the primers. It is important to note, however, that DNA polymerase doesn’t know where to start without the primers!
The final product is two exact copies of the original DNA template. Each daughter DNA contains one strand from the old template, and one brand new strand.
I went in depth on DNA replication here because this is the fundamental process that PCR is based on. For the sake of completeness, I will also briefly mention the other cellular machines that are responsible for manufacturing RNA and proteins.
(1) RNA polymerase will read certain chapters of the DNA manual, and write this information into RNA strings.
(2) Ribosomes will take the RNA strings, and use these instructions to build proteins.
Tying it all together: how does PCR work?
As described at the beginning of this article, PCR stands for “Polymerase Chain Reaction”. You can probably guess what “Polymerase” might refer to!
PCR is a procedure used by scientists to mimic the DNA replication that typically occurs in cells. There are certain ingredients that are absolutely required, some of which are shown in the figure below.
Remember helicase, primase, and DNA polymerase? We’ve only got one member of this troupe participating in PCR, which is DNA polymerase. Both helicase and primase are missing.
- Since we don’t have any helicase, we need to heat up the DNA template in order to unzip it.
- Since we don’t have any primase, we need to add our own synthetic primers to tell DNA polymerase where to start.
During PCR, we will repeatedly heat up and cool down the whole reaction mixture. This is called thermal cycling. With each cycle, the number of copies of the DNA template will double.
So there we have it! PCR is a method used by scientists to make lots of copies of a DNA template. We do this by borrowing a piece of cellular machinery called DNA polymerase, and artificially supply the other necessary components in the process.
Why is PCR useful?
As mentioned previously, there are actually two extra flavours of PCR called RT-PCR and qPCR which are used for SARS-CoV-2 diagnostic testing. I will be going into more detail about these procedures in a future article. You’ll be glad that you understood basic PCR before getting to these more advanced topics!
PCR has many more uses beyond diagnostic testing for viruses. For example, PCR is an essential step when performing DNA sequencing, which is used to print the order of “A”, “C”, “G”, “T” characters in a particular DNA string. Given the DNA sequence, it is possible to computationally predict what kinds of RNA and proteins might be made from it.
I hope this article has helped you appreciate the information flow from DNA to RNA to protein, which occurs in your body billions of times every day. It is truly amazing that scientists can mimic the highly complicated process of DNA replication, through the humble method of PCR.
Until next time!
References
- Alberts, B., Johnson, A. D., Morgan, D., Raff, M., Roberts, K. & Walter, P. Molecular Biology of the Cell. (Garland Science, 2017).
Further Reading
- PCR covered in a series of videos by the Journal of Visualized Experiments (JoVE). Access to JoVE is free until June 15th,2020!
- PCR Basics, a comprehensive guide written by biotechnology company ThermoFisherScientific.
