Quantitative Real Time PCR (qPCR)

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0:00-0:55 | What is qPCR?
0:55-1:30 | What is PCR?
1:30-3:30 | What is needed for PCR?
3:30-5:14 | How does PCR work?
5:14-6:17 | How does qPCR work?
6:17-7:34 | Why is qPCR useful?

Quantitative PCR (qPCR) is a technology which simultaneously amplifies and measures DNA. It is sometimes referred to as real-time PCR or even quantitative real-time PCR. In Swedish we say that a beloved child has many names and that certainly seems to be the case here. In a nutshell qPCR works exactly as normal PCR, except it adds two additional elements: These are a fluorescent dye, i.e. something that can be tracked and a fluorometer, i.e. a tracker. These two additional elements allows us to measure the DNA we are amplifying as it is being amplified. To better understand the whole process let us first take a look at how conventional PCR works.

Polymerase chain reaction or PCR can be thought of as a DNA copy machine. PCR is in other words a technique to copy or amplify small segments of DNA. It is both relatively cheap as well as fast. It is also useful since several instances of genetic and molecular analysis require significant amounts of sample DNA. To better visualize the different ingredients of PCR we can think of it in terms of building a house. The first thing we need is the DNA template to be copied, i.e. the DNA we want to amplify. This would be the blueprint of the house. Second we need DNA nucleotide bases or dNTPs. These A, G, C, and T bases are the building blocks of DNA after all. It naturally follows then that these nucleotides are the construction materials from which we can build our house. Third we need an enzyme known as Taq polymerase enzyme which adds these nucleotides according to complementary base pairing of the DNA template. In other words the Taq polymerase is our builder Fourth we need primers which are short stretches of DNA that the Taq polymerase enzyme or the builder can attach to or in other words upon which the builder can start building. In our house example these primers act as a foundation upon which the house can be built. Fifth and finally we need a buffer to ensure the right external conditions for the reaction. This can be thought of as nice weather, making the building process easier. Putting it all together, we have a builder (Taq polymerase) that starts building a house (new copied DNA sequence) upon the foundation (DNA primer). This builder assembles the building material (DNA nucleotides) according to our blueprint (DNA template) while the weather is nice (buffer). Hopefully that helps to more easily remember what goes on in this process. Next let us take a look at how this entire process actually takes inside the PCR reaction.

The polymerase chain reaction can be divided into three main phases. First we have denaturing, taking place at 94-95 degrees Celsius, which breaks the hydrogen bonds forcing the double-stranded DNA to separate into two pieces of single-stranded DNA. Second we have the annealing stage, occurring at 50-64 degrees Celsius, where the primers can attach to their specific location and from which the Taq polymerase can start synthesizing or building two new strands of DNA. One for each DNA strand. Third, the extending stage at 72 degrees Celsius during which the Taq DNA polymerase synthesizes DNA turning the two original pieces of **single**-stranded DNA into two pieces of **double-**stranded DNA. These double-stranded DNA molecules contain one strand of “old” template DNA and one strand of “new” synthesized DNA. During this process the Taq polymerase adds complementary bases to the DNA template one by one. (A-T and vice versa, C-G and vice versa) This results in the DNA doubling for each completed PCR process. These 3 processes of thermal cycling as it is called is repeated 20-40 times resulting in a HUGE number of copies of the original DNA sequence in a relatively short time.

Now how is qPCR different from normal PCR. Well as I alluded to earlier, the main difference lies in the fact that qPCR allows us to measure the PCR reaction while it is still occurring. This turns the process from qualitative into quantitative meaning that we get real-time data that corresponds to the amount of amplified DNA. qPCR follows the exact same working principle as normal PCR but it marks the nucleotides used in DNA synthesis with fluorescent labels. This is what allows us to measure the increase in DNA as it is being amplified. So how is qPCR currently used?

Quantitative PCR is currently used to detect, identify and quantify microorganisms that cause diseases such as bacteria, viruses and fungi. The lab tests for Covid-19 were usually carried out by PCR and qPCR for instance. It can also be used to detect and quantify genetically modified organisms. In addition PCR in general is often used in forensic research to amplify pieces of DNA found at a crime scene to allow us to identify the culprit. These are just a few of the many uses of qPCR.