What Do Scientists Actually Do?: PCR

Hello, dear readers, and I’m sorry for the wait between entries! Job-hunting is a full-time job, and then I went on vacation. In any case, welcome to the first “What Do Scientists Actually Do?” entry of Immortal Amaranthe.

If I were honest (and snarky), I would say that what biomedical researchers actually do is pipette. A ton. And that would cover about 80% of what I am going to write about in this and future “What Do Scientists Actually Do?” entries, at least when I’m talking about what biologists do. What is pipetting? Using a pipette (shown below) to move small amounts of liquid around. And…that’s how you do a lot of experiments. Pipetting. So much pipetting.

Immortal Amaranthe Pipette

https://www.coleparmer.com/i/labnet-biopette-p3940-10-autoclavable-pipette-adjustable-vol-0-5-10-0-ul/2460102

Image description: a medium blue laboratory pipette with a tip

This is definitely true for the procedure I’m going to discuss today: PCR. Thankfully, I used to love pipetting, but from now on, I’m going to talk mostly about the theory behind PCR, as well as some of the history. Oh, and what PCR actually is.

PCR stands for “polymerase chain reaction”. “Chain reaction” is one of those phrases I kept hearing when I was growing up and never really understood, and was glad to finally hear what it really meant in college. In case you share my childhood curiosity, a chain reaction is a reaction whose products contribute to the continuation of the reaction, or even speed it up. So what is a polymerase? A polymerase is an enzyme that makes long chains of RNA or DNA. So polymerase chain reaction—at least in a laboratory setting—is a method for amplifying small amounts of DNA several orders of magnitude.

PCR has been around since the early 80s, but a major development in PCR technology came in 1985 when a DNA polymerase that was stable at extremely high temperatures—Taq polymerase—was synthesized in a laboratory setting. (Fun fact: it was initially discovered in bacteria that live at extremely high temperatures.) This was useful because high temperatures are required to cause DNA strands to uncouple from each other so they can be copied, so it is advantageous to have a polymerase that can work at similarly high temperatures. PCR became much more easily usable in 1991, when the heat cycler—a machine that can rapidly bring its contents to extremely high temperatures—was put into production. Since then, PCR has become a staple of most molecular biology labs.

The polymerase chain reaction happens in three stages: denaturation, annealing (binding of short DNA sequences called primers to the DNA to be amplified), and extension of the DNA strands. In an actual PCR experiment, each cycle consists of the three stages, and a given experiment can take anywhere between 35 and 80 cycles to complete (usually).

Here’s a diagram from my master’s thesis depicting the first few cycles of a PCR experiment:

Immortal Amaranthe PCR Diagram

Image description: a diagram of the results of first four cycles of PCR, depicting the exponential increase in copies of DNA each cycle

So the amount of DNA increases exponentially (for a while, until what is called the “pseudolinear” stage, when the reaction can no longer be 100% efficient). Needless to say, PCR is a quick and efficient way to get from a tiny amount of DNA to a very large amount of DNA.

There are many different variations on PCR, but that’s probably not very interesting. I’m guessing you’re probably thinking something more along the lines of “Okay, but why would a scientist want to take a tiny amount of DNA and amplify it to make tons of DNA?” Well, for one, to analyze it. That’s what I did in my master’s thesis.

The idea behind my thesis was to use a tool called a molecular beacon probe to identify small amounts of mutant DNA among a much larger amount of wild type (normal) DNA. In order to do this, I used a type of PCR that amplified more of one DNA strand than the other. DNA’s two strands are called “sense” and “antisense”, with the sense strand being the one that actually codes for a protein; molecular beacon probes bind to the sense strand. The probes work better when there are less antisense strands competing with them for binding to the sense strand, so I used a type of PCR that made more sense strands. When the probes bind to their targets, mutant pieces of DNA, the probes light up, and the experimenter can measure the amount of fluorescence that they give off.

So what was the point of my thesis? Why is it useful to use PCR and molecular beacon probes to identify a mutation? Well, the idea behind the thesis is that molecular beacon probes can be used to identify cancer-related mutations in a human blood sample. When a person has cancer, they may have circulating tumor cells in their blood, and a PCR-and-molecular-beacon-probe test would be able to identify the mutant DNA from the tumor cells even though there is also a large amount of normal DNA in the blood sample.

I hope that wasn’t too complex; I just really wanted to talk about my thesis. I’m very proud of it. A slightly less complicated application of PCR is forensics-related genetic fingerprinting. If you have ever watched any crime procedural show, you’re probably at least vaguely familiar with genetic fingerprinting; you may have heard a character say that someone’s blood was a match for what was found at the crime scene. Have you ever wondered how forensic scientists on the show (and in real life, for that matter) can tell such a thing from a drop of blood or a tiny amount of saliva? Well, the answer is PCR. Forensics experts isolate small amounts of DNA from the evidence and amplify it with PCR before analyzing specific sequences to compare the sample to DNA from a suspect or a DNA database. Similar techniques are used in paternity testing.

Like I mentioned earlier, many molecular biology labs use PCR. In fact, I’ve used PCR in cancer research in a context other than my thesis. During my first laboratory experience, I was tasked with doing PCR on samples from chronic lymphocytic leukemia patients before the DNA could be sequenced. The results of the DNA sequencing were then given back to me so I could look for mutations in a particular gene that is often mutated in solid cancers. (There weren’t any. I was disappointed, but such is science. Hypotheses are frequently wrong.)

PCR-based procedures are also used in the following procedures or experiments:

-Tissue typing (testing to determine whether or not a potential organ donor is a match to the patient who needs a new organ)

-Genetic testing for heritable diseases

-HIV testing

-Testing for antibiotic resistance in TB

-Early diagnosis of leukemia, lymphoma, and other cancers

I think PCR is a pretty cool technique. Yeah, that’s incredibly nerdy, but if I weren’t a nerd, I wouldn’t be running a science blog, would I?