Components of the Reaction Mixture
Performing a polymerase chain reaction requires assembling a specific mixture of components in a single reaction tube.
First, a template DNA is required—the specific material to be copied. Four types of deoxynucleoside triphosphates (dATP, dGTP, dTTP, dCTP) serve as substrates for the synthesis of new strands, acting as building blocks.
The main catalyst of the process is Taq polymerase, a specialized thermostable enzyme that is not destroyed by high temperatures. To ensure this polymerase operates at maximum efficiency, a buffer solution containing magnesium ions (Mg²⁺) is added to the medium, serving as a vital enzymatic cofactor.
A key element that determines the specificity of the reaction is the pair of primers. These are artificially synthesized short single-stranded DNA molecules, ranging from 20 to 30 nucleotides in length. Their primary task is to serve as "starts" to initiate enzymatic synthesis. Primers are strictly complementary to the 3'-end sequences of the amplified region located on both strands of the template DNA. The concentration of primers in the mixture must significantly exceed the amount of the initial template, and the distance between them ultimately determines the exact length of the newly synthesized fragments.
Stages of a Single PCR Cycle
The entire process takes place in a specialized automated instrument called a thermal cycler (or amplifier). This device cyclically changes and maintains preset temperatures, fully automating the reaction. A single thermal cycle lasts from 1 to 2 minutes and includes three consecutive stages:
- Denaturation (heating to 94 °C). Under the influence of high temperature, the hydrogen bonds of the double-stranded DNA helix break. The initial molecule unwinds, and its strands (3'–5' and 5'–3') completely separate.
- Annealing (52–60 °C). The temperature inside the instrument decreases, allowing short primers to find their targets and bind complementarily to the corresponding regions on the template DNA strands.
- Polymerization or Elongation (72 °C). The device raises the temperature again to the optimal level for the thermostable DNA polymerase to function. The enzyme attaches to the primer binding site and begins synthesizing a new strand, sequentially extending it from the 3'-end.
Dynamics and Capabilities of the Method
The efficiency of the polymerase chain reaction is based on the principle of exponential growth. During the very first cycle, only the initial copies of the target DNA region are produced. In the second cycle, the instrument repeats the denaturation, annealing, and elongation phases, resulting in the doubling of DNA fragments. Starting from the 3rd and up to the 20th cycle, a rapid exponential increase in the number of copies is observed. In just 20 cycles, more than one million ($10^6$) copies of the PCR product are generated in the tube. Using an automated thermal cycler makes it possible to obtain this massive amount of target DNA in just 1–2 hours.
In modern biology and medicine, this method addresses a vast spectrum of tasks. PCR is widely used to identify various mutations directly within the DNA structure. The method allows the target region to be amplified for any downstream studies.
In medical diagnostics, PCR is indispensable for detecting foreign genes during infections caused by pathogenic viruses and bacteria. Furthermore, the technology is actively used to establish genetic relationships between organisms and even to determine whether a specific gene belongs to euchromatin or heterochromatin.