Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is one of the most important and widely used techniques in molecular biology, revolutionizing research in genetics, medicine, and biology. This technique enables the precise and rapid amplification of specific DNA segments and finds applications in various fields, including medical diagnostics, forensics, agriculture, and scientific research. This article will provide a detailed introduction to the principles, components, stages, and applications of this method.
Principles of Polymerase Chain Reaction
PCR is based on the ability of DNA polymerase to synthesize a new DNA strand using a template and primer. DNA polymerase is an enzyme that can add nucleotides to an existing DNA strand, thus creating a complementary strand. PCR involves multiple cycles of thermal stages that are repeated sequentially, doubling the amount of target DNA with each cycle.
Main Components of the PCR Reaction
To perform PCR, specific components are required, which include:
1. DNA Polymerase:
This enzyme is used for synthesizing the new DNA strand. The Taq polymerase enzyme, extracted from the thermophilic bacterium Thermus aquaticus, is commonly used in most PCR reactions. This enzyme is suitable for the high temperatures of the PCR cycle due to its thermal stability.
Principle of PCR Operation:
The principle of PCR, similar to the DNA replication process, is based on the enzyme DNA polymerase. Taq polymerase catalyzes the synthesis of a new DNA strand from the DNA template. This enzyme is derived from heat-resistant bacteria found in hot springs and hydrothermal vents. Since these vents are located at depths in the ocean and near active volcanic sites, the enzymes from these bacteria, including DNA polymerase, are highly thermally stable, with the most active form of this enzyme functioning optimally at 70°C. It should be noted that at this temperature, most human enzymes become inactive.
Below are explanations of three scientific terms related to enzymes:
Thermal Stability: This refers to the enzyme's ability to maintain its three-dimensional structure and function at temperatures higher than the ambient environment.
Processivity: This describes the enzyme's ability to remain attached to the substrate until the reaction is completed.
Fidelity: This refers to the enzyme's ability to incorporate the correct molecule at the appropriate site. For polymerases, this results in the complementary molecule being positioned opposite each nucleotide.
Enzyme Activity:
One of the factors that make Taq polymerase suitable and ideal for PCR is its thermal resistance, as the temperature during PCR alternately increases to denature the DNA strands and initiate replication again. The activity of Taq polymerase significantly increases at 70°C, and at temperatures around 90°C or higher, the structure of the enzyme is preserved without loss of activity, returning to its active state when the temperature decreases.
The activity of this enzyme at different temperatures can be summarized as follows:
Temperature 70 (°C) 60 Nucleotides Synthesized per Second
Temperature 55 (°C) 24 Nucleotides Synthesized per Second
Temperature 37 (°C) 1.5 Nucleotides Synthesized per Second
Temperature 22 (°C) 0.25 Nucleotides Synthesized per Second
Another important factor regarding Taq DNA polymerase is its high processivity, allowing it to process fragments up to three thousand nucleotides long, and with slight modifications in conditions, this number can be increased to four thousand nucleotides. Additionally, a notable characteristic of Taq DNA polymerase is its fidelity and proofreading ability, with an error rate of one nucleotide per three thousand bases.
2. Primers:
Primers are short, single-stranded DNA sequences that bind to their complementary regions on the template DNA and determine the starting point for DNA synthesis. Proper primer design plays a crucial role in the accuracy and efficiency of the reaction.
Additional Explanation:
Taq polymerase can only synthesize a new strand in the presence of primers. Primers are oligonucleotides made of DNA that provide a free OH group for the enzyme and designate a starting point for synthesis. In other words, this starting point is the free hydroxyl group at the 3' end of the last nucleotide of the primer molecule.
The primers required for PCR are typically short segments of single-stranded DNA, generally about 20 nucleotides in length. These segments are designed to encompass the target region. Therefore, in each PCR reaction, two primers are needed to bind to each of the two strands of DNA, with the connection occurring through hydrogen bonds between the primer and template DNA. The primer binding to the template strand is called the forward primer, and the primer binding to the complementary strand is called the reverse primer.
3. Nucleotides (dNTPs):
Nucleotides are the building blocks of DNA, comprising adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP).
4. Template DNA:
The DNA sample that contains the target sequence for amplification.
5. Reaction Buffer:
The environment that provides optimal conditions for enzyme activity and DNA stability. This buffer typically includes magnesium ions, which are essential for DNA polymerase function.
Additional Explanation:
The activity of any enzyme is maximized under specific conditions. In fact, buffers help create and maintain these specific conditions throughout the reaction. The Taq polymerase buffer, typically at a concentration of 10X, includes Tris HCl, KCl, and usually MgCl2. Tris HCl helps stabilize the pH between 8 and 9.5. Two factors play significant roles in this buffer: the presence of potassium ions, which enhances the stability of primer binding to the template during annealing, and magnesium ions, which act as a cofactor for the polymerase enzyme to reach its highest structural activity.
Steps of the PCR Reaction
PCR consists of three main stages that are repeated in each cycle:
Denaturation:
In this stage, the sample is heated to 94-98°C to separate the two DNA strands. This step typically lasts 20 to 30 seconds.
Annealing:
The reaction temperature is lowered to 50-65°C to allow the primers to bind to their complementary sequences on the template DNA. This temperature is determined based on the primer design and typically lasts 20-40 seconds.
Extension:
The temperature is raised to approximately 72°C, which is the optimal temperature for Taq polymerase activity. In this stage, the enzyme adds nucleotides to the ends of the primers, synthesizing the new DNA strand. The duration of this stage depends on the length of the target sequence, usually requiring 30 seconds for shorter sequences (up to 1000 base pairs). Longer sequences require more time.
During sequential cycles (typically 25 to 35 cycles), the amount of target DNA increases exponentially, resulting in large quantities of the desired sequence by the end.
Applications of PCR
Some of the most common applications of Polymerase Chain Reaction (PCR) include:
Investigating and studying genes involved in diseases
Examining infectious disease factors
Appropriate medical diagnostics
Amplifying a fragment of a gene transcript or Reverse Transcriptase PCR
Identity verification
Quantifying transcript copy numbers of a gene using Real-Time PCR
Comparing transcript copy numbers of a gene against another using Semi-quantitative PCR
Disease Diagnosis:
PCR is a vital tool in diagnosing pathogens such as viruses (including the coronavirus), bacteria, and parasites. This technique is particularly effective for rapid diagnosis of infectious diseases.
Forensic Science:
In forensic science, PCR is used to analyze DNA from small samples such as hair, blood, or saliva. This method aids in identifying individuals and solving crimes.
Genetic Research:
PCR plays a key role in discovering and studying genes, investigating genetic mutations, and better understanding genetic diseases.
Biotechnology and Molecular Biology:
PCR is used for gene cloning, studying gene expression, and producing DNA for various purposes.
Agriculture:
In the agricultural industry, PCR is applied to identify plant diseases, genetic modification, and detecting genetically modified organisms (GMOs).
Advantages and Limitations of PCR
Advantages:
High sensitivity: Capable of amplifying DNA from very low sample quantities.
Speed: The PCR process can be completed within a few hours.
Versatility: Usable in a variety of applications.
Limitations:
High sensitivity to contamination, which can lead to erroneous results.
Requires specific equipment such as a thermal cycler.
Limitations in amplifying long DNA segments.
Types of PCR
By making slight modifications to the components or temperature conditions of PCR, we can optimize this reaction for various purposes. Some of these include:
qPCR or Real-Time PCR
Multiplex PCR
Nested PCR
High Fidelity PCR
Fast PCR
Hot Start PCR
GC-Rich PCR
Long-range PCR
Arbitrary Primed PCR