Diagnostic Methods in Biotechnology: PCR
Discover the fundamental role of the Polymerase Chain Reaction (PCR) in molecular biology, as well as its impact on rapid medical diagnostics and the emergence of cutting-edge biotechnology innovations.
PCR, short for Polymerase Chain Reaction, is a technique widely used in molecular biology and biotechnology. As the name suggests, it refers to a chain reaction that occurs with the help of a specific enzyme called polymerase.
The purpose of PCR is to analyze a DNA molecule.
As is well known, DNA consists of nucleotides such as A, T, C, and G.
Each DNA molecule is a polymer formed by the arrangement of these nucleotides in a specific order.
If we have information about this sequence, we can use that information to identify the DNA molecule.
A Practical Example: Diagnosis of Tuberculosis
For example, the bacteria that cause tuberculosis in humans are called M. tuberculosis.
The DNA sequences of these bacteria that is, their nucleotide sequences, are known.
So, we can use PCR to determine whether this bacterium is present in a sputum sample taken from a patient.
To do this, we first extract the bacteria’s DNA from the sputum sample taken from the patient, and then use PCR to determine the nucleotide sequence of that DNA.
However, as is the case with this example, since the DNA of living organisms is quite long, sequencing the entire sequence would be an unnecessary effort.
This is because many regions of this DNA sequence may show similarities to those of many other bacteria and even other organisms.
For this reason, identifying only the DNA sequence specific to tuberculosis, rather than the entire DNA sequence, is a more practical approach.
To this end, that specific region of tuberculosis (for example, a region 300 nucleotides long) is amplified using PCR.
How Does the PCR Process Work?
So how is this region amplified?
Since the DNA sequence of this specific region, which is limited and unique to tuberculosis, is already known, short oligomers (called primers) that are complementary to the beginning and end of this specific region are used.
If you add the primers and the DNA to a reaction mixture containing the polymerase enzyme and the nucleotides A, T, G, and C, the PCR reaction mixture is ready.
When we place this mixture into a device called a thermal cycler, it becomes possible to produce numerous copies of the tuberculosis-specific DNA region using PCR (i.e., polymerization).
To perform polymerization, the “cycling” settings of the thermal cycler must be configured. For this purpose, three temperature steps are used: denaturation, annealing, and elongation.
During denaturation, the thermal cycler raises the temperature to 95oC. This causes the DNA we added to the PCR reaction to transition from a double-stranded to a single-stranded state, because the hydrogen bonds holding the two strands together break at this temperature.
During the annealing step, the temperature is lowered to a lower range, such as 55–65oC, so that hydrogen bonds between the two strands can begin to reform.
However, during this step, the large number of primers added to the reaction compete with the DNA’s original strands.
In other words, the primers bind to the beginning and end of the sequence specific to tuberculosis because they are complementary to it. To put it another way, the DNA’s own main strand cannot pair with itself; instead, it pairs with the primers.
During the elongation phase, the polymerase enzyme recognizes these primers and adds nucleotides in sequence to their ends, matching the complementary strand of the DNA.
This region of tuberculosis DNA is thus amplified in both directions by the polymerase enzyme.
This cycle produces a number of copies equal to the previous cycle raised to the power of 2 each time.
Consequently, after 30 cycles, theoretically 1 billion copies are produced from a single DNA molecule.
The amount of DNA in a sample taken from a patient is insufficient for analysis.
However, as a result of PCR, the target region in this small number of DNA molecules is amplified to billions of copies.
This may be sufficient for analysis.
After this stage, the analysis is performed using another technique called electrophoresis.
That’s a topic for another article...
Advantages and Disadvantages of PCR
Advantages of PCR:
Of course, PCR provides highly sensitive results because even a single copy can be amplified to billions of copies after 30 cycles.
For example, compared to diagnostic processes that rely on culturing bacteria, as is the case with tuberculosis, it is much faster. Normally, culturing the tuberculosis bacterium on standard LJ media takes 21 days, but with PCR, results can be obtained within hours.
Disadvantages of PCR:
It may be possible to determine whether the DNA obtained is from tuberculosis, as in our example, but it cannot tell whether the bacterium is alive or dead. Therefore, additional tests may be required depending on the situation.
Although not directly related to the technique itself, depending on the level of care in the laboratory where the work is conducted, the large number of DNA molecules produced may serve as a source of contamination for subsequent studies and could lead to erroneous results.
From Nobel Prize to Modern Biotechnology
PCR, which earned Dr. Kary Mullis the Nobel Prize in Chemistry in 1993, was invented by him. This revolutionary discovery played the largest role in the emergence of the most significant advancements in molecular biology and biotechnology. During the COVID-19 pandemic that emerged worldwide after 2019, the PCR technique also played the most significant life-saving role in diagnosing the then-unknown pathogen.
At Rapdi Biotech, we continue to widely use the PCR technique, which is commonly employed in all areas of molecular biology and biotechnology, in our SELEX cycles for aptamer selection.
References:
Ma, T. (1995). Applications and Limitations of Polymerase Chain Reaction Amplification. CHEST, 108(5), 1393-1404.
Mullis, K. B. (1993). Kary B. Mullis – Nobel Lecture. NobelPrize.org. Nobel Prize Outreach.
Khehra, N., Padda, I. S., & Zubair, M. (2025). Polymerase Chain Reaction (PCR). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing.
Disclaimer: The information provided in this article is for educational and informational purposes only, based on current scientific literature. It does not constitute medical advice, diagnosis, or treatment. For any medical concerns or diagnostic needs, always consult with a qualified healthcare professional.