Reaction mixture components
To perform the polymerase chain reaction, a specific mixture of components must be assembled in a single test tube.
First of all, the initial DNA template is required — the very material to be copied. Four types of deoxynucleoside triphosphates (dATP, dGTP, dTTP, dCTP) are used as substrates for the synthesis of new chains. They act as building blocks.
The main catalyst of the process is Taq polymerase, a special thermostable enzyme that is not destroyed by high temperatures. To make this polymerase work as efficiently as possible, a buffer solution containing magnesium ions (Mg²⁺), which serve an essential function as an enzymatic cofactor, is added to the medium.
The key element that defines the specificity of the reaction is the two primers. These are artificially created short single-stranded DNA molecules, 20 to 30 nucleotides in length. Their main task is to serve as a "seed" for the initiation of enzymatic synthesis. The primers are strictly complementary to the 3'-terminal sequences of the amplified region located on both strands of the template DNA. The concentration of primers in the mixture must greatly exceed the amount of the original 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 special automated device called a thermal cycler (or amplifier). This apparatus cyclically changes and maintains the specified temperatures, fully automating the reaction. A single temperature cycle lasts from 1 to 2 minutes and includes three consecutive steps:
- Denaturation (heating to 94 °C). Under the influence of high temperature, the hydrogen bonds of the double-stranded DNA helix are disrupted. The original molecule unwinds, and its strands (3'—5' and 5'—3') completely diverge.
- Annealing (52–60 °C). English term — annealing. The temperature inside the device 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 level that is optimal for the operation of thermostable DNA polymerase. 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 specified DNA region are formed. In the second cycle, the device repeats the phases of denaturation, annealing, and elongation, resulting in the doubling of the number of DNA fragments. Starting from the 3rd and up to the 20th cycle, a rapid exponential growth in the number of copies is observed. In just 20 cycles, more than one million ($10^6$) copies of the PCR product are formed in the test tube. Using an automatic thermal cycler allows obtaining this huge amount of studied DNA in just 1–2 hours.
In modern biology and medicine, this method allows solving a huge range of tasks. PCR is widely used to identify various mutations directly in the DNA structure. The method allows amplifying the target region for any further research.
In medical diagnostics, PCR is indispensable for detecting foreign genes during body infection with pathogenic viruses and bacteria. In addition, the technology is actively used to establish genetic relatedness between organisms and even to determine whether a particular gene belongs to euchromatin or heterochromatin.
Briefly: answers to topical questions
What is the difference between classical PCR and Real-Time PCR?
Real-time PCR differs in that sources describe it as a quantitative method: it evaluates the viral load in logarithms.
| Characteristic | Classical PCR | Real-Time PCR |
|---|---|---|
| Main principle | Amplification: multiple copying of a gene or a specific DNA region in vitro | Real-time PCR; quantitative method |
| What is evaluated | Obtaining a large number of copies of a gene or DNA fragment | Viral load in logarithms |
| Clinical significance according to sources | Identification of mutations, foreign genes during infection, obtaining a DNA region for further research | Differentiation of clinically significant HPV amounts and minor amounts |
What medical and biological tasks can be solved by the PCR method?
The PCR method allows solving the following tasks:
- Identify mutations in the DNA structure.
- Diagnose infections and identify foreign genes during infection of the body with viruses and pathogenic organisms.
- Diagnose hereditary diseases.
- Obtain a DNA region in quantities sufficient for further research.
- Clone genes.
- Identify identity in forensic medicine.
- Establish genetic relatedness of organisms.
- Establish whether a gene belongs to eu- or heterochromatin.
Abbreviation of steps: DAE — Denaturation (disruption of the helix), Annealing (primer binding), Elongation/polymerization (chain synthesis).
Class and exam questions
What is the function of Taq polymerase?
This is a thermostable enzyme that extends new DNA strands starting from the 3'-end of the primers. Due to its stability, it is not destroyed by high temperature during denaturation.
Why are magnesium ions (Mg²⁺) needed in the reaction mixture?
Magnesium ions are part of the buffer and act as an obligatory cofactor necessary for the correct and efficient operation of DNA polymerase.
What determines the length of the copied DNA fragment?
The length of the synthesized region strictly depends on the distance between two primers that complementarily bind to opposite strands of the DNA template.
How long does it take to get millions of copies?
Thanks to the thermal cycler, one cycle takes 1–2 minutes, and the entire amplification process yielding more than a million copies (about 20 cycles) is completed in 1–2 hours.
What else to study on this topic
- The mechanism of complementary binding of primers to the template.
- The role of magnesium ions and other cofactors in enzymatic catalysis.
- Differences in the structure and functions of euchromatin and heterochromatin.
- Types of deoxynucleoside triphosphates and their participation in DNA synthesis.
- Principles of automation of genetic research using thermal cyclers.
Master the entire topic in the "Biochemistry" course
This is a summary. The course covers everything tested in classes and exams.
- Complete study guide on "Polymerase chain reaction" and 3,616 more pages of the subject
- Assignments and quizzes after every topic — just like in a colloquium
- AI tutor answers questions on the topic anytime
- 115 video lessons on the subject
Other topics in the section "Template Biosyntheses"
Free video lessons: biochemistryShort lessons with study notes — no registration required.
Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.