Principle of the method and purpose
PCR is the primary diagnostic tool for identifying bacterial and viral infections. The method is based on the geometric growth of the number of copies of the target DNA region (doubling in each cycle).
The process requires primers — short oligonucleotides that act as "starters". They are fully complementary to the 3' ends of the gene to be amplified. The main advantage of PCR is the ability to work directly with the test material (clinical samples, water, food products), bypassing the lengthy stage of isolating a pure culture of the pathogen.
Mechanism of the amplification cycle
The entire process proceeds cyclically inside a special device — a thermal cycler, which strictly controls temperature changes. Each cycle consists of three sequential steps:
- Denaturation (92–95 °C). Upon heating, hydrogen bonds break, and the initial double-stranded DNA template molecule separates into two distinct strands.
- Annealing (37–60 °C). The temperature decreases, allowing primers to find their targets and bind complementarily to specific regions at the 3' ends of both single-stranded templates.
- Elongation (63–75 °C). The mixture is heated to the optimum temperature for thermophilic DNA polymerase. This enzyme captures free nucleotides and synthesizes new DNA strands, moving outward from the primers.
As a result of a single cycle, two new double-stranded copies of the gene are formed. Next, the cycle starts over from the denaturation stage of the newly synthesized molecules.
Real-time PCR
This is a modern automated modification of the classic method. Its key feature is the simultaneous occurrence of DNA accumulation (amplification) and detection processes.
A specific molecular probe is added to the reaction mixture. When it binds to the synthesized DNA strand, a fluorescent signal is generated. Based on the fluorescence intensity, the instrument assesses the amount of product in real time.
Advantages of the modification:
- Express diagnostics: results are ready in 20–60 minutes.
- Ultra-sensitivity: detects even single RNA or DNA molecules.
- Automation: minimization of manual labor.
Real-time PCR is actively used not only for qualitative detection, but also for precise quantification of viral load, as well as for molecular strain typing.
Briefly: answers to topical questions
What components of the reaction mixture are necessary to perform PCR?
Five main components of the reaction mixture are required to perform the polymerase chain reaction. The mixture includes:
- DNA template — the test fragment serving as the basis for copying.
- Substrates — four types of deoxynucleoside triphosphates (dATP, dGTP, dTTP, dCTP).
- Primers — two artificially synthesized short single-stranded DNA regions acting as primers.
- Enzyme — thermostable Taq polymerase, which carries out the synthesis of the new strand.
- Buffer — a solution containing Mg²⁺ ions as a cofactor.
What is the most frequently used thermophilic DNA polymerase in PCR called, and from which microorganism is it isolated?
The provided sources indicate thermostable Taq polymerase for PCR. Information about the microorganism from which it is isolated is not provided in the sources. The enzyme participates in the elongation stage: it synthesizes the complementary DNA strand starting from the primer; the optimal temperature for Taq polymerase is specified as 72 °C.
What method is used to detect amplification products in conventional PCR?
The provided sources do not specify the general method for detecting amplification products in conventional PCR. It is only indicated that in Real-time PCR, amplification and detection occur simultaneously due to a special molecular probe generating a fluorescent signal. Additionally, the description of RFLP analysis following PCR amplification mentions DNA restriction, gel electrophoresis, and detection using radioactively labeled oligonucleotide probes, but this is described for that specific assay rather than as a general method for conventional PCR.
PCR steps are easy to remember by the acronym ADE: Denaturation (separation) → Annealing (primer attachment) → Elongation (strand extension).
Class and exam questions
How does the amount of DNA change during the reaction?
The amount of the target DNA region increases exponentially — strictly doubling after each completed cycle.
What is the difference between real-time PCR and conventional PCR?
In real-time PCR, amplification and product detection occur simultaneously due to the fluorescence of molecular probes, and the entire process is automated.
What else to study on this topic
- Structure and properties of thermophilic DNA polymerase
- Features of optimizing primer annealing temperature
- Assessment of viral load by quantitative method
- Application of PCR for molecular typing
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