So far in our little series, we’ve covered how genetic material is extracted from a swab, and how a very basic PCR works.
This third part will cover how the theories of a basic PCR are then applied to the fancy type of PCR used for COVID testing…
Which is called real time reverse transcription (RT)-PCR.
Or real time RT-PCR/RT-PCR for short.
So if you remember anything from the previous post on basic PCR, just remember that PCR is about amplifying, or making lots and lots of copies of the same template genetic material.
You start off with a small amount of sample/template, and by the end of your PCR run, you end up with an exponential increase in the number of copies.
The same overall theory still applies for RT-PCR, but there’s extra steps and ingredients required to do this.
Plus you need a much, much fancier thermocycler.
Because now, we’re using light, or fluorescence.
Amongst other fancy things. 🧐
Okay, so let’s just begin by recapping the basic ingredients/components required to run a PCR. You’ll need:
- Template genetic material (basic PCR uses DNA)
- Primers (the short, complementary strands of DNA that binds to your template – determines specificity/target)
- Polymerase (The thing that extends your copy strands to match the template)
- Buffer and dNTPs (I’m combining these because that’s how most PCR kits work)
Now, for an RT-PCR, you’ll need some changes and additions.
You still have all of the above, but firstly you also need:
- A fluorescent probe
This usually comes with the primers if you buy a PCR kit. Much like the primers, they’re designed to bind to your template. Not overlapping the same area as the primer (otherwise they would be competing for the same spot), but a region a little further down. It just has to be before where your reverse primer would bind.
Because otherwise it would be outside of the area being copied, right?
I’ll explain why this fluorescence is important a little later.
In addition to the fluorescent probe, there’s also another enzyme.
Because if you remember from part 1, COVID’s genetic material isn’t DNA.
RNA is similar to DNA, but it does have some differences. I won’t go into detail here, but essentially, they’re too different to treat in the exact same way as DNA.
But wait- isn’t your PCR designed to work using DNA as the template…?
Well, I suppose you can try to work with RNA and try and swap everything out to be RNA instead of DNA.
But RNA is a bastard to work with. They degrade very easily, in that it can’t withstand environmental stresses in the same way as DNA.
You’ll still have residues of RNA, but the strand would have fallen apart into tiny pieces if you don’t keep things at optimum conditions. Not particularly helpful if you want a strand to copy.
I suppose it’s like a literal jigsaw puzzle. If you try and pick it up, and do anything with it- it falls apart. You’ll still have the pieces, but you won’t know what the picture was anymore. So you have to glue it together, put it against a stronger board, etc. so that it doesn’t. Same thing with RNA. You have to keep it cold, keep it in the right buffers, keep it away from enzymes that degrade it, etc. so that it remains intact. 😅
One of my personal traumas from my Ph. D days is trying to isolate RNA from the bacteria I was working with. F*** me it was difficult!! It just disappeared without a trace during the whole process. I hated it.
Anyway- to avoid all that stress and trauma, the best (and easiest) thing to do for your RNA COVID sample is to just change the material entirely into something more… stable.
And that’s exactly what this enzyme does.
It takes an RNA template, and then literally copies it into DNA. It’s the same sequence, in essence, but it’s now in DNA form, which is much more robust and stable. Like transcribing into different languages. The meaning is still the same, but it’s just in a different format.
This copy DNA is called complementary DNA (cDNA). And it denotes any piece of DNA that’s been copied from an RNA template.
This process of converting RNA into DNA is what’s called reverse transcription, which is where the assay gets its name from.
So this enzyme, naturally, is called a reverse transcriptase. Because it reverse transcribes as its sole function. 😅😂
FYI transcription itself is the process of converting DNA into RNA. We do that all the time in our bodies (especially when making proteins), but we can’t do reverse transcription- that’s what viruses do, because they’re weird.
*ducks for cover from the angry Virologists*
And finally, there’s also an additional step during RT-PCR in that the polymerase can cleave, or chop up the fluorescent probe mentioned earlier, during the extension phase.
Why that’s important will be covered further down.
So an RT-PCR tube needs the following things:
- Template genetic material (RT-PCR uses RNA)
- Primers and probes (both bind to template in a complementary manner)
- Reverse transcriptase (converts RNA template into cDNA)
- Polymerase (still a DNA polymerase like the basic PCR, because by this point everything is cDNA)
- Buffer and dNTPs (because the end product is still DNA, this part is still pretty much the same)
I’ve highlighted the changes from a basic PCR, but as you can see, it’s still very similar.
Now it’s time to dive into each step and explain what’s happening.
Step one. Reverse transcription
This is the new step required in an RT-PCR.
You have to first use your reverse transcriptase to copy your RNA template into cDNA.
I’ve already explained this bit above so I’ll leave it at that. 😂
The time it takes for a reverse transcriptase to reverse transcribe will depend on the enzyme itself, but for the standard tests used against SARS-CoV-2, it can be anything from 15 minutes to 30 minutes.
And yes, there are primers involved in this step as well, so that the reverse transcriptase knows which region to convert into cDNA. They’ll be targeting the exact same area throughout this entire RT-PCR run (including in the annealing step during cycling).
But now, you no longer have RNA- it’s all cDNA from here on out. No need to worry as much about your template disappearing without a trace.
Step two. Denaturation.
This is identical to the basic PCR. The two strands of cDNA come apart at around 95 degrees.
Step three. Annealing and Extension.
Okay, this is where it gets a bit more complicated.
Compared to a standard PCR, where the annealing and extension steps are distinct (in temperature and time), the RT-PCR for COVID testing combines these steps into one.
But more to the point- this is where that fluorescent probe comes into play.
As I mentioned, the probe can bind the cDNA target, similar to a primer.
Let’s zoom in on this probe.
As you can see, the probe includes a reporter (R) and quencher (Q) portion.
Both the reporter and quencher are physically attached to the probe.
For a fluorescent probe, the reporter (R) is the portion that releases fluorescence (light). The quencher (Q), in turn, quenches this fluorescence from the reporter, but only when they’re in close proximity of one another.
Here’s a diagram to show this.
So as you can see, while the reporter and probe are within a short enough distance of one another (i.e. the length of the probe), the quencher can smother the reporter from emitting light.
But as soon as the reporter is free and far enough away from the quencher- you get light. 💡
Still with me?
Now, I mentioned earlier that the polymerase is able to chop up the probe during the extension phase.
Well, when the polymerase starts to copy and extend the cDNA strand, it hits the probe because it’s in the way.
And because the polymerase just needs to extend the cDNA, it doesn’t give a s*** that there’s a probe in its way, because… it’ll just chop it up.
The probe is completely displaced, the quencher is regretting its life decisions, and the reporter is free to release all the light that it can. All the while the polymerase continues to make cDNA
Step four. Cycling (repeat steps two to three).
And then you just repeat this whole process over and over again.
And you can imagine, with each iteration, the number of copies of cDNA increases- as does the number of free, unquenched reporters, because eventually they all get released by the polymerase during the extension phase.
So the more copies of cDNA there are, the more light being emitted.
This will be important for part 4, when I go through the thermocycler/RT-PCR machine and how these results are detected and interpreted.
Just as a bonus content, this is what the cycle parameters might look like on an RT-PCR thermocycler program.
You can see the reverse transcription phase (55 degrees for 20 minutes), then initial denaturation (outside of cycling) at 94 degrees for 2 minutes, before it goes into the cyclic denaturation (94 degrees for 15 seconds), annealing and extension (60 degrees for 30 seconds) for a total of 40 times.
You can also input how much sample volume is in each tube (25 microlitres here), as well as an estimated run time (1:29:00, so just under 90 minutes).
All of these conditions are specified by the enzyme kit and primer and probe kit, because the manufacturer’s have rigorously tested their products to figure out what conditions work best for their stuff.
But if you were doing everything in scratch, then you would have to go through a process of trial and error to figure out what works best. That’s fun (not).
A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬
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