So part 1 covered the process of extracting genetic material from your samples.
But before I can explain the next step in the COVID PCR testing process, I first have to explain what a PCR is. 😅
PCR in a nutshell is the process of making lots and lots of copies of specific genetic material.
You start off with a small amount of genetic material (your template), and by using enzymes and other bits and pieces I will describe shortly, you make lots of identical copies. Exponentially.
That’s the core concept of a PCR.
But obviously there’s a bit more to it than that, so I’ll describe all the things needed to do a PCR and how it works below.
If you didn’t know this already, PCR stands for ‘polymerase chain reaction‘.
You’ll be forgiven if you thought it was pipette, cry, repeat.
I even have a shirt that says it!
The basic components of your most standard PCR (not a COVID one) are as follows.
In a single reaction tube you need:
- Template genetic material
- The starting genetic material, which could be your test sample, or your positive control
- A standard, basic PCR uses DNA
- Primers (forward and reverse)
- These are short sequences of DNA that determine what specific area you’ll be copying
- This is the enzyme (because it ends in ‘ase’) that helps create the copy strands
- dNTPs (nucleotides)
- the Gs, As, Ts, and Cs that make up DNA
- Buffer mix
- Helps the reaction run smoothly by maintaining optimum conditions for your enzymes and genetic material
- Might need to add additional chemicals provided in the kit (according to manufacturer’s instructions)
- Most kits these days have the dNTPs already in the buffer mix
But just having these in a tube won’t do much at all.
So how do you perform a PCR?
By using a thermocycler, or a PCR machine.
They may look pretty fancy (because they are), but what these things are essentially designed to do is to cycle through different temperatures for a set amount of time.
Because a PCR only works if you put the reaction at very specific temperatures for a very specific length of time. 😅
So let’s go through the basic steps of a PCR below.
Step 1. Denaturation
Hopefully you’ve all seen DNA before. It’s double-stranded, it forms a helix… all that jazz.
And they’re made up of building blocks called nucleotides. For DNA, there are four kinds.
Adenine (A), thymine (T), cytosine (C), and guanosine (G).
Each of these letters can be ordered and arranged in a number of ways, and the unique sequences form the information (genes) required to build proteins. This is the central dogma of biology.
But for the purposes of today, all you need to remember is that the nucleotides are what form DNA (which is double stranded), and that the ordering is very specific and unique for each gene.
Also, each nucleotide (base) has a complementary base that they will bind to.
A binds to T (and vice versa), and C binds to G (and vice versa). These are depicted by the dotted lines in the above figure.
In order to make more copies of DNA, the first step in the PCR process is to separate out the two strands of template DNA in a process known as denaturation.
What you need to do is to heat the reaction tube containing your PCR components to around 95 degrees Celsius.
This heat causes the two strands to separate, because it weakens the bond between the complementary bases. If you heated it up even higher, then even the single strands would start to break apart- but we’re not going to do that. We just want the two strands to separate apart.
This initial denaturation step might last a couple of minutes at most. If a protocol has an additional denaturation step at the very start that goes for 5-10 minutes, this is used predominantly to break open whole viral particles or cells. If you’ve already extracted the genetic material (i.e. there’s no extra barrier between the genetic material and the enzymes), then you don’t have to worry about this additional denaturation step too much.
Step two. Annealing
Now that the strands are separated, it’s time for the second step of the PCR.
The annealing step brings in the next character of our PCR. Primers.
Primers are short sequences of DNA (~18-30 bases long) that have been designed to bind certain sequences of your template. Primers determine what gene segment you’re amplifying.
This requires prior knowledge of your template sequence, i.e. you have to know what sequence of bases your template is made up of.
Given that most genes (the bits that carry all the vital info to make proteins) is longer than 18-30 bases, the primers target specific regions of genes. It could be the start, it could be the middle, or it could be near the end. Designer’s choice.
Now, I said these primers bind to the template DNA. This again uses complementary base pairing.
Say your template DNA had a sequence,
Well then, in order to bind that bit of DNA, you need to design a primer that’s going to read,
Because, again, As bind to Ts, and Cs bind to Gs. Having a complementary sequence means your primers will bind to that template DNA.
Now, remember how I said that you need a forward and a reverse primer?
Well, that’s because DNA (and RNA) is directional. You can actually see it in the above diagram.
The start end is the 5′ (‘five prime’) end.
The tail end is the 3′ (three prime’) end.
Can you now see that the two strands of DNA are running in opposite directions? The top strand runs left to right, while the bottom strand runs right to left.
So naturally, when you design primers for a PCR, you need primers for both strands. That’s why there’s a forward primer and a reverse primer. To account for both of these strands and the directions they run in.
Anyway, the primers anneal (stick to the template DNA) at around 50-60 degrees. The annealing temperature is entirely dependent upon the specific primers you use, so you might need to change the temp every time you use a different set of primers.
Not sure what your annealing temperature is? There’s a mathematical formula for calculating it by hand, but these days, we all just use online calculators. 😂 We just use the ‘melting temperature’ result after we plug in the sequence we might like for our primers (also called oligonucleotides or oligos), and then add or remove bases depending on whether it’s too low or too high a temperature. Both your forward and reverse primers have to have either the same or plus or minus 2 degrees temperature difference from each other, because if they’re too different, they won’t anneal to the template in the same reaction tube.
Too complicated? Don’t worry- if you buy a primer kit, the manufacturer’s will tell you what temperature to use. 😂 Just follow their instructions.
Step three. Extension
Okay- so you’ve got your template strands, they’ve separated during the denaturing step, and now they’ve got primers attached during the annealing step.
The third step is called extension, and it’s when the polymerase (in pale blue) extends this new copy strand of DNA to match the template (as a complement), as accurately as possible.
The polymerase can’t just extend strands on its own- it needs the building materials to do so, as well.
That’s where the dNTPs come into play. They’re the actual bases that get incorporated into the new strand. It’s kind of like this- the polymerase is the brickie, and the dNTPs are the bricks (comes in four different colours!!).
The time it takes for extension to occur will depend on the length of your template and the speed at which your polymerase will make new strands. All of that will already be tested by the manufacturer of the polymerase, so just follow their instructions. 👍🏼
Now, extension occurs at 70 degrees Celsius.
If you’re someone who thought- why the hell would you ever need to elongate DNA at 70 degrees?? That’s super hot!
Well, you’re a very observant cookie. 🍪
Our polymerase in our bodies would’ve inactivated long, long ago, because our polymerases are designed to work at around 37 degrees (i.e. human body temp).
But the polymerase used in PCRs aren’t from humans, or mammals.
They’re usually from weird, bacteria-like organisms called Archaea, who generally live in really crazy, or extreme environmental conditions. Sometimes they’re halophiles (that love salt). Sometimes they’re found in underwater vents that spew sulphur and magma.
Either way, they’re called extremophiles, because they live in (and like) extreme environmental conditions.
So you can imagine, sometime ago, some scientist found an Archaean that grew at 70 degrees, whose polymerase worked at 70 degrees, and thought… that’s weird.
And then later, someone else went-
I’ve got a use for that.
The process of DNA replication, that’s been found in another organism, has been hijacked and used in an artificial setting.
That’s science in nutshell, really. 😅
But why even bother with such a weird polymerase? Why not use a mammalian one? Or any other normal organism? Why does the reaction have to be at such a high temperature?
Well, there’s a few reasons why.
The template strand, primers, complementary template strand, and the polymerase all have to work together in harmony for a PCR to work.
First the temperature needs to be high so that the primer can bind its template DNA in lieu of the complement (the Other strand).
But then there’s also the fact that the high denaturation temp can eventually inhibit normal (37 degrees) polymerases- because, lo and behold, normal polymerases don’t do so well being put at 90+ degrees (let alone repeatedly). This was the problem with using polymerases from E. coli. They just couldn’t keep up with the repeat exposure to high temperatures.
And then there’s also the problem of non-specific binding of the primers. Sometimes, if the relationship is a bit cool, the primers go off to find other partner/s. 😅
I.e. If the reaction temperature is a bit cooler than what the primer wants, then the primer will bind random template DNA- not in the region it’s supposed to.
So, with all of these things in mind, the polymerases used in PCR were selected because they can withstand repeat exposure to high temperatures, so it kinda solves these problems.
The other important thing for polymerases is fidelity. They have to be able to make a complementary copy of the template strand as accurately as possible. Low fidelity enzymes make occasional mistakes (a T instead of a G, a G instead of an A, etc.). Those mistakes can build up, and especially if you’re after 100% accuracy… they become a bit of a problem. High fidelity enzymes make significantly less mistakes, but they’re also very expensive. 😅
The general rule of thumb is, if your purpose is just to screen, or to check whether a specific sequence of DNA is present- then the low fidelity polymerase is fine.
But if you’re after 100% accuracy, then you need to splurge a bit more and use something with more fidelity.
Unrelated to PCR, but if you’re curious about how DNA replication happens in our own bodies, it’s a little more complicated, but there’s a lovely TED talk by Dr Drew Berry from the Walter and Eliza Hall Institute (WEHI) below.
Step four. Repeat steps one through to three.
Okay, so you’ve successfully made a copy from each of the two strands? Great!
Now we need to repeat the whole cycle again.
Usually between 20 to 45 times.
But eventually, you go from one copy of double stranded DNA to two, then four, then 16, and so on and so forth. And suddenly you have an exponential amount of DNA. Given that you’re never going to be starting with one copy of DNA (usually in a sample you’ll have multiple template copies to start you off), the final copy number can be bajillions. That’s a word.
The number of cycles (i.e. the number of times you repeat steps 1-3) will depend on the following:
- The amount of dNTPs available to make new copies of DNA
- How much stamina your enzyme has
Obviously if you run out of bricks, you can’t keep building your wall.
If your brickie becomes exhausted, then they’re not going to be building more wall.
All of that will have been tested by your enzyme manufacturer, so you can just follow their instructions as well. 😅
So those are the basic steps involved in a PCR. There’s denaturation (separation of strands), annealing (attachment of primers), extension (elongation of the complementary copy DNA strand by polymerase), then repeat.
Hopefully you were able to kind of follow along. 😂
But now we’ve covered the basics, we can then apply it to the PCR testing used for detecting COVID.
This is a little bit different to ya basic PCR described here, but the underlying theory is still the same.
Stay tuned for part 3!
A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬
Yes, I was “able to kind of follow along”, with stress on the “kind of”. Thanks for the explanations.
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