When faced with the task of describing the world around us and the depth of human experience, the limited vocabulary of the English language seems wholly insufficient. In an attempt to rectify the language’s shortcomings, I’ve invented the following words. I share them here along with some examples of usage, and I urge you to include these new terms in your lexicon.
Tinger (noun) – the intense frustration that comes when someone is unable or unwilling to recognize something that is incredibly obvious. Ex: “I am overwhelmed with tinger when my uncle talks about evolution being a sin; not believing in it, the concept itself.” –
Macont (noun) – the aroma of horse urine and automobile exhaust found near horse-drawn carriages in urban environments. Derived from French or something. Ex: “When you can smell the macont, you know you’re in Central Park.” –
Flemp (adjective) – the quality of being not quite as good as remembered. Ex: “I really liked this drive-through soup restaurant when I first moved to the city, but tonight it was a bit flemp.” –
Oosay (noun) – the urge to look at one’s reflection every five minutes after getting a haircut. Ex: “I was so overcome with oosay when I stepped into the restroom, I didn’t notice the police tape.” –
Skoince (verb) – to recognize and avoid an acquaintance in a public place before they notice you. Ex: “I almost ran into Ricky at the supermarket, luckily he was preoccupied by the Natural Light display and I was able to skoince.”
Those of us who have been following the news over the past year and a half1 have likely become accustomed to hearing the terms “PCR” or “PCR test” all the time. Specifically, you might have heard that a PCR test is the gold standard for detecting viral infections because it is so sensitive, but why is that? And what even is a PCR? And why are my socks wet? These are all very good questions, and luckily I am here to answer them for you (except that last one, you’re on your own with there).
Firstly, I think it makes sense to explain what the letters “PCR” stand for before we go any further, just to get it out of the way. “PCR” stands for Polymerase Chain Reaction. We don’t really need to know what this means because it will become clear later. For now, it is enough to understand that PCR is simply a technique that scientists use to amplify the amount of a specific piece of genetic material, i.e. DNA. To understand how this works, we first have to know a bit more about DNA and how it works.
DNA, as some of you may know, is an acronym that stands for DeoxyriboNucleic Acid. More significantly, DNA is the basis of almost all life as we know it2 and contains the genetic information that makes us into the organisms that we are. Most people are familiar with the following depiction of DNA as a double-helix shape that looks like a twisted ladder.
the quintessential picture of a strand of DNA
While this picture is not incorrect, it doesn’t really help us to understand how the molecule of DNA actually works. Instead, I think a more suitable way to visualize it would be the following:
a ladder with a very specific set of rungs
There is something noticeably different (I hope) in this image – namely, the “rungs” of the ladder are marked with pairs of letters. By now, those of you who took biology in school might know what I’m getting at with this image, but for those who didn’t, fear not – I will elaborate. The “rungs” here are the so-called bases of the DNA, made up of four unique molecules – A,T,C,G (these initials come from the chemical names of the molecules: Adenine, Thymine, Cytosine, and Guanine). The rungs of the ladder are formed by one base from each side pairing up with another base on the opposite side; G always pairs up with C, and A always pairs up with T, in a consistent pattern. It is the specific sequence of these bases – i.e. the order in which they are arranged on the ladder – that determines the genetic information carried by the DNA. When you hear someone talk about a “DNA sequence” or the “genetic code”, this is what they’re talking about.
So now we know a little bit about how DNA carries the information necessary to make life. In fact, a reasonably short strand of DNA can carry a huge amount of information – the mathematically inclined among you might recognize that for a strand of DNA with N number of bases, there are 4N number of unique combinations. For reference, the human genome is about three billion bases long, which gives an incomprehensibly large number of unique combinations (granted, most of our DNA contains general, species-level information like number of eyes, teeth, limbs, organs, etc. The amount of DNA that makes you unique from another human person is considerably smaller, but still huge).
This knowledge about the genetic code is all well and good and dandy, but to really understand how PCR works, there are a couple more things that we need to understand about our new favorite genetic molecule. First in this category is the fact that DNA has two strands, arranged like so:
DNA with two strands, arranged in a very specific way.
“But millibeep” I hear you lament, “we know that DNA has two strands, we saw it in that beautiful picture from a few moments ago.”
Correct, but that picture was still missing a key piece of information. In this drawing, we can see the two strands that make up our stretch of DNA, arranged into a top strand and a bottom strand. Because the bases of each strand always pair up in a specific way (G with C, A with T), the top strand and bottom strand carry the same information as reverse images of each other (one consequence of this is information redundancy – if one strand is damaged, the other strand maintains the information so it can be repaired properly).
Furthermore, you might have noticed the labels on either end of each strand, which look like 5′ and 3′, but are pronouced “five-prime” and “three-prime”. There are technical reasons why they are named this way, but for now the important thing is to know that the two ends of a given strand are different. When a DNA molecule is formed, the top and bottom strands are always oriented opposite to one another, with the 5′ end on the top corresponding to the 3′ end on the bottom. Therefore, to have the complete set of information about how the DNA is arranged, we need to know not only the sequence of the bases, but their orientation with respect to the ends as well.
This end-to-end orientation is important when it comes to replicating a strand of DNA, something that our own cells do every day, and a crucial process to the concept of PCR. When a polymerase (the biological machine that replicates DNA) makes a copy of a strand of DNA, two things happen. First, the double stranded structure is partially “unzipped” to expose a region of single-stranded DNA. Second, a polymerase will “see” the unfinished, single-stranded DNA and rush to the scene. There it begins copying the DNA from the 5′ end towards the 3′ end, and only in this direction. Due to its nature, the polymerase’s machinery is only capable of functioning in this one direction.
a hard working polymerase notices the unfinished molecule and begins to diligently copy the strand of DNA
Alright, we are almost ready to understand what PCR is and how it works, I promise. But first, we need to know just one more thing about DNA.
Like all molecules, DNA is held together by energetic bonds between its parts. The strength of these bonds is determined by the exact chemical structure, but the important thing to know is that the bonds between the bases are weak enough that they can be melted apart with the right amount of heat. This is just like an ice cube melting into water as the thermal energy breaks the bonds between its molecules. Also like the ice cube, once the thermal energy is removed, the bonds can re-form and the bases will connect back together (in genetics, this process is referred to annealing, rather than freezing, but the concept is the same).
DNA can melt apart and re-form with the addition and subtraction of thermal energy
Alright, now that we know:
a) how DNA is structured, b) how a polymerase copies DNA, and c) how DNA can melt apart and re-form,
we are finally ready to see how PCR actually works!
As I said earlier, PCR is a technique used to amplify the amount of a specific piece of DNA in a sample, i.e. to increase the number of copies of that piece many, many times (the keen-minded among you might already be imagining how the polymerase comes into all this). In the case of testing for a viral infection, the PCR will be amplifying a specific part of the virus’ genetic material.
In order to amplify a specific piece of DNA, it is predictably necessary to know the sequence of that piece (lucky for us, DNA sequencing technology has come a long way!). Once we know the sequence of the piece we want to amplify, we need to make short, single-stranded pieces of DNA called primers that have the same sequences as part of the target sequence. These can be created synthetically and the process for this is interesting, but too complicated to include here. For each piece of DNA we want to amplify, which I will now call the target sequence, we need two primers – one for the top strand and one for the bottom strand (respectively called “forward” and “reverse” by convention), like so:
By now, you may be able to guess why these short strands are called “primers”; they will be used to “prime” the chain reaction that gives the process its name. Let’s go through it step by step.
To start off, we have our initial sample that contains a mixture of our double-stranded target sequence as well as many, many copies of our single-stranded forward and reverse primers, and our polymerases.
a thrilling mixture of double-stranded target sequence, polymerases, and single stranded primers
Then, using a piece of lab equipment called a thermocycler, we heat the mixture just enough so that the double-stranded target sequence will melt apart. During the heating, nothing really happens to the primers since they are already single-stranded.
at just the right temperature, our target sequence will melt apart into single-stranded DNA
After melting apart, all of the bases of the target sequence are wide open and ready for anything – they’re single and ready to mingle. Taking advantage of this, we cool the mixture back down enough that some of the bonds will start to anneal, or reconnect. Because we have included many, many copies of our forward and reverse primers, chances are that some of the primers will anneal to the target sequence before the original strands can reattach to one another.
before the two strands can find each other to anneal, the primers swoop in and attach themselves
At this point, we will have some long strands of the target sequence that are partially double-stranded because of the primers, but not completely! This means that we have unfinished DNA on our hands – luckily, we have a guy for that. The polymerase will jump in to attach itself to the unfinished strand created by the primer, and begin to extend it by copying the target sequence to create a piece of double-stranded DNA.
the polymerase cannot abide unfinished DNA, and will extend the primer to create a single-stranded piece
If this process is able to occur on both strands of the original target sequence, once the polymerase(s) are done copying, we will have two copies of the original DNA – one made from the top strand and one made from the bottom strand. Because we added many, many copies of the primers to the mix, all we have to do is repeat the process over and over again (hence the “cycler” part of the word “thermocycler”). At each repetition, the number of copies of the original target sequence will increase exponentially – one becomes two, two become four, and so on (hopefully it is clear now where the name “Polymerase Chain Reaction” comes from). This means that in a relatively short time, we can increase how much target sequence DNA we have by a huge amount!
PCR will exponentially increase the amount of target DNA
The exponential increase of target DNA is the strength of this technique – in a few short rounds of thermal cycling, the amount of our target can be exponentially increased to an easily detectable level (for the curious, this is usually on the order of a few micrograms per milliliter). This is what makes PCR so powerful for detecting, say, the sequence of a novel bat virus – all you need for a positive result (in theory) is a single copy of the genetic material3,4.
And there we have it, exactly what PCR is and how it works. I hope that I was able to demystify this concept for you and that you’ll now be slightly more informed when writing letters-to-the-editor or shouting from soapboxes or drafting legislation or whatever it is you people do when not reading my articles.
As a final note, I debated with myself about whether to include an explanation here about how the final product of the PCR (the hugely amplified amount of target DNA) is detected. It is an interesting, and (I think) fairly easy to understand process. However, I ultimately decided that will be the subject of a follow-up article, when I can actually demonstrate the technique in the lab (with pictures!).
Until next time.
/millibeep
Footnotes: 1 – If you haven’t been following the news, don’t worry; nothing significant whatsoever has happened and you can go back to whatever it is you were doing.
2 – Some life forms, like viruses, use a slightly different genetic molecule called RNA.
3 – As an aside, this is much more sensitive than a typical viral antigen test, which tests for the presence of the viral antigens (the parts of the virus that your immune system reacts to). By their nature, antigen tests have a fairly high detection threshold – meaning that a sample needs a large number of antigens to register a positive result.
4 – In case anyone is alarmed at the idea of making many copies of a virus’ genetic material, fear not. It is safe because the PCR reaction would only target a small fraction of the overall genetic code – far less than is necessary to make a functioning virus. In fact, the greater danger comes from handling the samples of patients’ spit.
It seems like a straightforward question at first, but if we scratch the surface just a little bit the enormous complexity of attempting an answer becomes apparent. However, since hubris is my middle name, I remain undaunted and can promise my beloved readers a complete answer over the next couple dozen paragraphs.
The first complication we encounter is the fact that we do not currently have a good definition of what life actually is.
“Hang on there, ol’ beep ol’ pal,” I hear you say, “what do you mean we don’t have a good definition? Life is obviously the state of being alive and not-dead.”
Firstly, how dare you address me in such a fashion. Secondly, technically you are correct, but only technically. If we define “life” in that way, we very quickly run into the fiendish circular logic trap of defining what it means to be dead (can something that never lived be dead?) and alive (the opposite of dead? the state of having life?). You can see how we may tie ourselves in knots and not in a fun way.
There have been many attempts to define life and all of them seem to be incomplete in one way or another. Persons who paid attention in middle and high school biology classes may remember learning that there are seven criteria for life, as follows:
Homeostasis – the regulation of processes to maintain an equilibrium
Organization – being composed of at least one cell, so called the “basic unit of life”
Metabolism – transforming energy into cellular components (usually chemically)
Growth – expanding oneself through metabolizing resources, i.e. “a higher rate of anabolism than catabolism” (from wikipedia) in order to increase the size of its components
Adaption – the ability to undergo evolution in response to the environment
Stimulus response – the broad term for reacting to aspects of the environment (different from number 5)
Reproduction – the ability to make more copies of oneself, usually described as sexual or asexual
To the first order, this list is a good approximation of what biologists consider to be properties of living things, but it starts to fall apart when we consider the interesting edge cases. Obviously, humans fit all of these criteria and most humans reading this are arguably alive. This also applies without much room for dissent to most mammals, plants, fish, and insects that one might come across outdoors. The interesting arguments happen when we apply this to the more interesting organisms.
One fairly well-known debate with regards to the above list has to do with whether viruses can be described as alive or not. Some people make the argument that because viruses are not capable of replicating outside of their host cells, they cannot be fully alive and instead are merely machines to replicate their own genome. Those on the other side would say that because we can apply the principles of Darwinian evolution to viruses, they must be considered to be a form of life. I am of the opinion that viruses are living things, for two reasons.
The first reason is beautifully illustrated by the name of my favorite class of viruses: phages. These are a class of viruses that infect bacterial (and sometimes viral) cells. The psychology and classic language students among the audience will recognize that the word “phage” comes from the Greek word meaning “to devour”. They were so named because they were initially thought to be “eating” the bacterial cultures in which they were first observed. To me, this is an excellent refutation to the argument that viruses are not alive because they cannot replicate without their host cells. To a phage, a host cell is merely a resource, like nutrients to a bacterium. In my view, it would be just as incorrect to say that because a human cannot replicate without food, they are not alive. The second, and far simpler reason is that viruses can be killed (think alcohol based hand-sanitizers, heat, etc.), which I maintain is a solid heuristic argument, if a bit circular.
As an aside, there is an article by Eugene Koonin and Petro Starokadomskyy in the journal Stud Hist Philos Biol Biomed Sci (which might be my favorite abbreviated journal title ever) that suggests a completely different view to the debate. Rather than viewing viruses as being alive or not, Koonin and Starokadomskyy suggest it is more useful to think of viruses in terms of their position on the selfishness-cooperativity axis, along which all biological replicators sit.
By now, we’ve gotten a bit off track. This article is not about viruses and whether or not they are alive, its about the simplest form of life that we could successfully argue for. So far, all we’ve done is muddy the waters with viruses and talk about how the list of criteria for life is not completely useful. With that in mind, it is worth noting that the definition of life that NASA uses (according to wikipedia) is much simpler: “a self-sustaining chemical system capable of Darwinian evolution.” This definition is decidedly broader and may be more useful for our purposes.
For all the discussion about viruses and whether they are alive and should have rights and freedoms under the law, they are not the simplest form of life, despite being exceedingly simple. In fact, there are three other examples of arguably simpler forms of life that I can think of. Let’s take a look at them one at a time.
Viroids. The first example I can think of as being simpler than a virus is the aptly named “viroid”. Like a virus, a viroid is a small amount of genetic material (i.e. RNA) that can infect other cells to replicate itself. Unlike viruses, however, the viroid lacks a protein shell that protects a virus’ genome during transmission and helps it infect cells. Instead, the viroid is essentially just a loop of RNA that uses its host to replicate itself. Basically all of the same arguments about viruses apply to viroids, and certain classifications may not even distinguish between them. That said, they go on the list because, without the protein shell, they are decidedly simpler than viruses.
Ribozymes. Like viroids, ribozymes are molecules of RNA. The difference here is that a ribozyme is an RNA molecule that is capable of some enzymatic activity (in fact, the name comes from RIBOnucleic acid enZYME). Those of you who recall your biology courses may remember that the molecules that replicate your genetic material (polymerases) are also enzymes. It has been hypothesized that a ribozyme structured in just the right way would be able to replicate itself. While this hasn’t precisely been observed yet, Tracey Lincoln and Gerald Joyce were able to produce a pair of ribozymes that replicated each other. This candidate gets points off for technically existing only in theory (for the moment), but it gains those points back for how nicely it fits into the RNA-world hypothesis.
Prions. The third and final candidate in this list for simplest life form is also the one that will likely cause the most consternation among biologists. In short, a prion is a misfolded protein that is able to induce similar proteins to misfold in the same way. This has significant effects if it happens with any of the proteins we need to live, and is the cause of diseases like Mad Cow and Scrapie (terrifyingly, the section about treatment of prion diseases on wikipedia is very short and begins with the sentence: “There are no effective treatments for prion diseases.”). The main difference between a prion and the other entries on this list is the prion’s lack of genetic material. Instead, it is information about how the protein misfolds that gets transmitted.
This begs an interesting philosophical question: if it is a form of life, is it the prion itself, or the information about how to misfold that is actually alive. If we start classifying information as being alive, we may be obliged to include much of the universe’s phenomena to the point where the label becomes useless. On the other hand, there are likely some people who take a thermodynamic view of life and would argue that the information (genetic or otherwise) is the only defining feature of life to begin with. The whole debate could devolve into a moment reminiscent of Diogenes’ plucked chicken (“Behold, a man!”).
We could spend lifetimes chasing our tails around this question but since this would be a waste of our time and tails, I would rather take the viewpoint that any strict labeling system will break down if you prod the edges enough. In any question of philosophy (and life overall) we must remember the old adage that labels were made for (hu)man, not (hu)man for the labels. We use terms like “alive” and “dead” to help make sense of the universe as we try to describe it to ourselves. The moment that our understanding reaches a point where the labels we use are no longer useful (or, indeed, more of a hindrance than a help), they should be cast aside to make way for new descriptions and ways of thinking.
Anyway, I hope you learned something from reading this, and I think we can all agree that the award for simplest form of life goes to prions. Definitely prions.
/millibeep
Author’s note – This post has more links to wikipedia than I normally include. For those that are unaware, wikipedia is a non-profit that runs off volunteer work and donations. If you use it a lot, like I do, please consider setting up a regular monthly donation of a couple bucks. It helps people all over the world with access to high-quality, encyclopedic knowledge and makes it possible for people like me to write articles like this one.
Please go get vaccinated! If you are medically eligible, getting vaccinated against Covid-19 is the most proactive and effective thing you can do to help end the global pandemic.
For the US residents, find valuable information here.
While all things must be considered critically, one of the groups of people who are generally trustworthy are the doctors and scientists who are dedicated to saving peoples’ lives in a once-in-a-century pandemic.
Also, you can already be tracked by your smartphone.
millibeep 