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Polymerase Chain Reaction (PCR)

Polymerase chain reaction, PCR

For medical students2 min readUpdated 2026-10-10

Polymerase Chain Reaction (PCR) is a technique for the in vitro amplification (multiple copying) of a specific microbial or human gene. It allows for the rapid detection of target DNA sequences in any sample without requiring prior isolation of a pure culture.

Core PrincipleGeometric amplification of the DNA quantity with each thermal cycle.
Main AdvantageDoes not require obtaining a pure culture of the microorganism.
EquipmentA thermal cycler (amplifier) for precise temperature modulation.
Real-time PCRAnalysis of the sample takes only 20–60 minutes.

Principle and Purpose

PCR is a primary diagnostic tool for identifying bacterial and viral infections. The method is based on the geometric growth of the number of copies of a target DNA region (doubling in each cycle).

The process requires primers—short oligonucleotides that act as molecular 'starts.' They are completely complementary to the 3' ends of the gene to be amplified. The primary 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 occurs cyclically inside a specialized device called a thermal cycler, which strictly controls temperature changes. Each cycle consists of three sequential steps:

  1. Denaturation (92–95 °C). Heating disrupts hydrogen bonds, separating the original double-stranded DNA template into two individual single strands.
  2. Annealing (37–60 °C). The temperature is lowered, allowing primers to locate their targets and bind complementarily to specific sequences at the 3' ends of both single-stranded templates.
  3. 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 one cycle, two new double-stranded copies of the gene are formed. The cycle then restarts with the denaturation step applied to the newly synthesized molecules.

Real-Time PCR

This is a modern automated modification of the classical method. Its key feature is the simultaneous progression of DNA accumulation (amplification) and detection.

A specific molecular probe is added to the reaction mixture. When it binds to the synthesized DNA strand, a fluorescent signal is generated. The device assesses the amount of product in real time based on the intensity of the fluorescence.

Advantages of this modification:

Real-time PCR is actively used not only for qualitative detection but also for precise viral load quantification and molecular strain typing.

Mnemonic

The PCR steps can be easily remembered with the acronym D-A-E: Denaturation (separate) → Annealing (attach primers) → Elongation (extend strand).

Frequently asked questions

What components of the reaction mixture are required for PCR?

Polymerase chain reaction requires five core components in the reaction mixture. These include:

  • DNA template — the target 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 sequences acting as starting points.
  • Enzyme — a thermostable Taq polymerase that synthesizes the new strand.
  • Buffer — a solution containing Mg²⁺ ions as a cofactor.
What is the most commonly used thermophilic DNA polymerase in PCR, and from what microorganism is it isolated?

The provided sources specify the thermostable Taq polymerase for PCR. Information regarding the specific microorganism from which it is isolated is not mentioned in the text. The enzyme participates in the elongation stage: it synthesizes the complementary DNA strand starting from the primer; the optimal temperature for Taq polymerase is stated as 72 °C.

What method is used to detect amplification products in conventional PCR?

The provided sources do not specify a general detection method for amplification products in conventional PCR. It only notes that in real-time PCR, amplification and detection occur simultaneously via a specialized molecular probe generating a fluorescent signal. Additionally, RFLP analysis following PCR amplification employs DNA restriction, gel electrophoresis, and detection with radiolabeled oligonucleotide probes, but this is described for that specific analysis rather than as a general method for classical PCR.

How does the amount of DNA change during the reaction?

The amount of the target DNA region increases in a geometric progression—it strictly doubles 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.

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