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?

The History of Science: Dr. Rosalind Franklin

Well, it looks like it’s time for my first entry! Let’s talk about one of my favorite scientists: Dr. Rosalind Franklin.

Dr. Franklin’s name often comes up in high school biology classes when students are first learning about the discovery of DNA’s structure. I would be willing to bet that several of the students in those classes are already familiar with the duo Watson and Crick when they first hear the name Rosalind Franklin and see Photo 51. If you ask those who study biology at higher levels about Rosalind Franklin, you might see their face screw up in anger and hear them snarl that Watson and Crick—or you might hear the name Wilkins—stole Dr. Franklin’s work. You might also hear that Dr. Franklin wasn’t given the credit she deserved due to sexism. Still others might say that the only reason Dr. Franklin is said to not have gotten sufficient credit for her work is because she died prior to Drs. Wilkins, Watson, and Crick being awarded the Nobel Prize, or that Dr. Franklin didn’t actually realize her results indicated that DNA’s structure was helical.

So what’s the real story? Was Dr. Franklin’s work stolen? If she had such definitive results on the helical structure of DNA, why didn’t she publish it before Watson and Crick did? I had to do more research than I expected to find out the answers to these questions, and in this entry, I plan to share those answers with you, dear readers.

But first, I want to share some information on Dr. Franklin herself. As I mentioned above, she passed away before the Nobel Prize for the discovery of DNA’s structure was awarded. She died tragically young; she was only 37 when she succumbed to ovarian cancer. She was born in the United Kingdom in 1920 into a Jewish family. According to Dr. Franklin’s sister, the family supported her scientific endeavors despite the fact that the sexism that exists in STEM fields today was even more rampant in the past.

While most people think of DNA as related predominantly to biology—it is occasionally referred to as “the molecule of life”—Dr. Franklin was primarily a chemist. While her contribution to the study of DNA was recognized posthumously, Dr. Franklin was still a celebrated scientist during her life. Her study of the porosity—the percentage of empty spaces in a material—of coal, which was the subject of her PhD thesis, provided critical information on how different types of coal would perform as fuels. She was a PhD student during World War II, and not only did her work with coal contribute to the design of gas masks with charcoal filters, but she also volunteered as an Air Raid Warden.

Dr. Franklin also did important work on the structures of RNA and viruses. After her work with DNA (which I will discuss later), she made groundbreaking discoveries on the structure of tobacco mosaic virus. Tobacco mosaic virus, often called TMV, is what is termed a model organism: a living thing that provides useful information for biologists on many similar other living things…and is usually easy to study. Dr. Franklin’s discoveries that the proteins in TMV were arranged in a spiral and that the RNA in TMV resides inside a groove in the protein arrangement were crucial to the understanding of several other similar viruses. Dr. Franklin was at the top of her game, cranking out papers on structural virology at an impressive speed, when her life was cut short.

In case you’re impatiently tapping your foot while waiting for me to talk about DNA, dear readers, I’m about to get to it. Dr. Franklin started working at King’s College London in 1950. She had done extensive work with a technique called X-ray crystallography—a method for determining the structure of a material that can form a crystal—after earning her PhD. She was hired to do similar work on proteins, but the department head reassigned Dr. Franklin to study DNA fibers. Dr. Franklin was able to use her past experience with X-ray diffraction to improve the quality of the results her department’s experiments were yielding. She discovered that DNA had two forms, which she called “A” and “B”. She noted that DNA fibers were long and thin when wet (form “B”), and shorter and fatter when dry (form “A”).

Some suggest that Dr. Franklin never realized that DNA was helical, which is why she did not publish that discovery ahead of Watson and Crick. The truth is that Dr. Franklin was a perfectionist who wanted to make absolutely sure that she had correctly interpreted all her data. In lecture notes dated November 1951, Dr. Franklin wrote: “The results suggest a helical structure (which must be very closely packed) containing 2, 3 or 4 co‐axial nucleic acid chains per helical unit, and having the phosphate groups near the outside.” In 1952, asymmetrical images of the “A” form of DNA caused Dr. Franklin to balk at the idea of claiming that both forms were helical. By 1953, she had reconciled all of her data and became convinced that both “A” and “B” forms of DNA were helical. Not only did she now know that DNA was helical, but she had deduced that DNA was a double helix: two helices intertwined with each other like a twisted ladder. She began work on several manuscripts on the subject, and one day before Watson and Crick completed their model of “B” DNA, two of Dr. Franklin’s papers on “A” DNA’s structure reached the headquarters of a journal called Acta Crystallographica. Years later, another manuscript dated March 1953 was found in Dr. Franklin’s files, this one discussing the structure of “B” DNA.

I don’t know about you, dear readers, but I’m pretty convinced that Dr. Franklin discovered the helical structure of DNA independent of Watson and Crick.

You know what, I’m not done gushing about Dr. Franklin’s research. It’s also important to note that not only did she figure out that DNA was a double helix, but she also knew that DNA contained a sequence of nitrogenous bases: key parts of the building blocks of DNA. She also deduced that the sequence of bases provided the genetic code. She even discovered that the nucleotides—the aforementioned building blocks of DNA—were paired with each other. I’m sorry, but that’s impressive. Actually, no, I’m not sorry.

“But what about the controversy?” you might be saying. “Did Watson and Crick steal her work?” Well, all right, dear readers. Let’s talk about the controversy. In 1952, Dr. Franklin’s student, Raymond Gosling, took one of the most famous photographs in science: Photo 51, an image of the “B” form of DNA.

Here it is.
Photo 51.png

https://en.wikipedia.org/wiki/Photo_51#/media/File:Photo_51_x-ray_diffraction_image

Image description: an X-ray crystallography image in grainy black and white showing small horizontal black bars in an “X” shape on a circular gray field

The crux of the controversy is this: Dr. Watson had the epiphany that DNA was helical when he saw Photo 51, which he saw without the permission of Dr. Franklin (or Raymond Gosling). In my research for this entry, I stumbled across at least one person claiming that Watson and Crick would have discovered the helical structure of DNA with Dr. Franklin’s consensual help had Dr. Watson been paying attention during a seminar Dr. Franklin gave on the relative distances of repetitive elements in DNA. (Dr. Watson admits to this inattention in his book The Double Helix: A Personal Account of the Discovery.) However, Photo 51 was taken after this lecture, and regardless of speculation, Dr. Watson had his realization when a colleague of Dr. Franklin’s—Dr. Maurice Wilkins—showed him Photo 51, again, without Dr. Franklin’s permission. I’m pretty certain that at no point in recent history was showing your labmates’ work to competing scientists considered anything but unscrupulous. Well, unscrupulous if not a terrible idea, since it could lead to you getting scooped. (“Scooped” is what scientists call it when someone else publishes results you’ve been working for before you do.)

Why in the world would Dr. Wilkins do this? That isn’t clear. What is clear is the sequence of events: on January 30, 1953, Dr. Watson arrived at King’s College London, bearing a manuscript containing another researcher’s incorrect model of the structure of DNA. He was looking for Dr. Wilkins, who wasn’t in his office, and Dr. Watson instead went to Dr. Franklin’s lab and insisted that they all collaborate before the author of the incorrect paper realized his model was wrong. As part of his insistence, Dr. Watson suggested that Dr. Franklin didn’t know how to interpret her own data, and Dr. Franklin was (understandably, I think) irritated. As he was scurrying away from this confrontation, Dr. Watson ran into Dr. Wilkins, who tried to console him and then showed him Photo 51. Some theorize that Dr. Wilkins did this because he had a terrible relationship with Dr. Franklin, who intimidated him; Dr. Wilkins was shy and retiring. Still others theorize that sexism was a factor in Dr. Wilkins’ actions. I can’t figure out people’s motivations when they aren’t historical figures that I’ve only read about, so I don’t know what Dr. Wilkins was thinking, but I do know he was in the wrong.

At no point was Dr. Franklin aware of her influence on Watson and Crick’s work. In modern science, if someone helps you with your research, you acknowledge their contribution in your paper. In April of 1953, Watson and Crick published their model for the structure of “B” DNA in Nature—a very, very reputable journal—without properly crediting Dr. Franklin. Later in the same issue, several articles by Dr. Wilkins and Dr. Franklin appeared in support of Watson and Crick’s model.

So was Dr. Franklin’s work stolen? I wouldn’t go that far. Was she not given enough credit? Definitely. Watson and Crick are still seen as the primary discoverers of DNA’s double helical structure when Dr. Franklin’s work was just as if not more important, and Watson and Crick likely would not have been able to publish that Nature paper without Photo 51. Was sexism a factor in Dr. Wilkins’ decision to show Photo 51 to Dr. Watson? I think that is possible; it was the 1950s, and as for anyone who doesn’t believe that there has always been sexism in science and that it still isn’t prevalent, I would like to know what life is like on their planet. (I mean, while Dr. Franklin was working at King’s College London, the female scientists were not allowed to eat in the common room. Also, if you read The Double Helix…wow, Watson and Crick have no idea how sexist and arrogant their attitudes were. No idea.) More important than what was in the heads of Drs. Watson, Crick, and Wilkins, though, is the fact that Dr. Franklin still isn’t given enough credit. To this day. I can’t show statistically significant data proving that sexism is a factor in that, but I would still bank on it.

If you take anything away from this entry, dear readers, is that Dr. Rosalind Elsie Franklin was an incredible, brilliant scientist who made many groundbreaking discoveries and who deserves all the credit in the world. Oh, and that the answer to “is sexism a factor in this?” is going to be “yes” 99% of the time if it involves women in science.