What is Real-Time PCR?
By Lauren Bambusch
When speaking with food safety managers trying to upgrade their testing methods, we are often asked —what is real-time PCR? How does it work? The quick answer is: PCR (or polymerase chain reaction, for those science nerds out there) is a technique for multiplying a specific strand of DNA or RNA millions of times by manipulating the cell's natural machinery. Once multiplied or amplified, the target DNA can readily be detected though a variety of methods.
But how does it work? PCR, as it was developed in its modern form by Dr. Kary Mullis in the 1980's, is a fairly simple process on its face—take a strand or segment of DNA, add a pinch of building blocks of life, a smidge of primers (small DNA segments that tell the cellular machinery ‘start copying here'), stir in some of cellular machinery (that's where the word polymerase comes in), and cycle between high and low temperatures 30-50 times, a process called thermocycling. Now a days we have instruments like InstantLabs' Hunter that automatically cycle between temperatures, allowing you to sit back and relax while the magic is working, but in the early days of PCR, thermocycling was done manually by grad students, who moved the small PCR tubes between hot and warm water baths every 30-60 seconds for a few hours (thank goodness I am too young to have ever had to do that myself).
Whether you're manually shuffling tubes or can kick back while an automated instrument does that for you, here's how the process works:
1. Mix ingredients
First, you'll need a sample with the potential DNA segment you'd like to verify. Any kind of DNA works for this—bacterial, animal, plant, human—at the most basic level, they're all the same. Second you'll need the building blocks of life; a buffer, cations, and most importantly, things called nucleotides, which create the genetic ‘code'. Also, you'll need the polymerase—this is your scribe, the machinery that chugs along the DNA strands, replicating the sequence as it goes. Lastly, you'll need some primers. These are short segments of DNA that can bind to a single-stranded DNA segment and give the polymerase something to hold on to as it starts to copy.
2. Heat the mixture up
DNA comes securely packed in a double helix formation—two strands that wrap around each other in a coil-like shape. This helps to protect and shelter the coded part of DNA, but at the same time serves to hide it away. In order to break that helix open, you add heat and voila, the double DNA strands un-curl and you have two single strands of to play with.
3. Cool the mixture back down
DNA isn't the only thing susceptible to heat. At these high temperatures the cellular machinery is not fully operational, but cool it back down and the primers begin attaching to the matching DNA segments so the polymerase can chug along making copies along the way.
4. Wait for a little bit
It doesn't take long—anywhere from 10-30 seconds and that polymerase will have made you a perfect copy of the DNA segment.
5. Repeat as needed
Need more than one copy? Just repeat the process until you have as much DNA as you need.
Each cycle doubles the amount of the target DNA that exists in the sample. As the cycling repeats, so does the doubling of the number of DNA segments. Due to the power of exponential growth, you quickly get a very large number of DNA copies; you'll have 2,048 copies after just the first ten cycles. After 30 cycles? You'll have over 1 billion copies. Yes, that's billion, with a “B”.
You may be wondering what you do with all those copies of DNA. You can do a lot things, but when you are hunting for a specific target of DNA, say a Salmonella bacteria, you also add little fluorescent tags into the mix, called probes. When you add energy in the form of a specific wavelength of light, the fluorescent part of that probe is excited, letting off a tiny burst of light in a different wavelength, kind of like colored Morse code. One little burst of light, or even 2 , 4 or 8, is too small for even the most sensitive instrument to detect.
But if you have a million copies? A billion? That's another story. As your instrument detects the light, it translates that into a ‘DNA FOUND HERE!' signal called relative light units (RLUs). The more DNA, the brighter the signal. That is what makes the classic PCR curve go up exponentially.
And that, ladies and gentlemen, is real-time PCR. In case you are interested, the duplication does not go on forever. Each replication uses up a bit of the building blocks added to the reaction; once those get used up, try as you might, the replication ends and the PCR curve flattens out. After all, you can't bake bread if you don't have any flour.
As you can imagine, because PCR is looking for an exact match between the target DNA and the primer, it can be very precise. Being able to turn one copy of DNA into billions allows PCR to be very sensitive. It is a great tool for detecting pathogens in foods—or any DNA—just about anywhere.
A discussion about the origins and applications of PCR in its many forms isn't complete without a nod to Dr. Kary Mullis, a brilliant and…ahem… interesting person. In fact, he attributes the invention of PCR to his long-term LSD use, saying in a 1997 interview, “What if I had not taken LSD ever; would I have still invented PCR? I don't know. I doubt it. I seriously doubt it.” Well, here's to Kary Mullis and the unconventional route of science. To find out more, pick up a copy of Mullis's book Dancing Naked in the Mind Field , which will give you a laugh-out-loud look into the mind of this fascinating and outspoken Nobel laureate.