The Polymerase Chain Reaction (PCR) is a foundational technique in molecular biology that generates millions to billions of copies of a specific DNA segment in a short time. This process essentially “photocopies” a tiny piece of genetic material in a test tube, driven by cycles of heating and cooling. PCR is widely used in medical diagnostics to identify pathogens, in forensic science for DNA fingerprinting, and in biomedical research to study gene function and variation. Setting up a successful reaction requires precise control over the ingredients and thermal conditions.
Essential Components for the Reaction
The reaction mixture must contain five core ingredients to successfully amplify the target DNA sequence. This mixture is usually assembled in a small, thin-walled tube designed for efficient heat transfer. The most fundamental ingredient is the template DNA, which contains the specific sequence a researcher intends to copy.
Two short, single-stranded DNA fragments called primers are also added to define the boundaries of the target region. These primers are designed to bind to the opposite ends of the desired sequence, providing a starting point for the copying enzyme.
The enzyme responsible for building the new DNA strands is a heat-stable DNA polymerase, most commonly Taq polymerase, isolated from the bacterium Thermus aquaticus. This enzyme can withstand the extreme temperatures necessary for the reaction. The polymerase requires deoxynucleotide triphosphates (dNTPs), which are the individual building blocks of DNA.
The four types of dNTPs—dATP, dGTP, dCTP, and dTTP—serve as the raw material for the new DNA strands. Finally, a reaction buffer is included to maintain a stable pH environment for the enzyme activity. The buffer also provides necessary ions, such as magnesium chloride (MgCl₂), which acts as a cofactor to enhance the Taq polymerase’s function, with an optimal final concentration ranging from 1.5 to 2.0 mM.
Calculating and Preparing the Reaction Mix
The physical setup begins with calculating the required volume of each component to achieve the desired final concentration in the total reaction volume, often 25 or 50 microliters. All work must be performed using aseptic techniques to prevent contamination from foreign DNA. Template DNA is added, 1–25 ng of genomic DNA or 1 pg–10 ng for plasmid DNA.
The forward and reverse primers are added to achieve a final concentration between 0.1 and 0.5 µM each, balancing specificity with reaction efficiency. A master mix is created by combining water, buffer, dNTPs, polymerase, and both primers. This approach minimizes pipetting errors and ensures all individual reaction tubes receive the exact same proportions of shared components.
The master mix is aliquoted into the individual PCR tubes. Template DNA is then added to each tube separately, often alongside a positive control (known DNA) and a negative control (water instead of template). Once combined, the tubes must be sealed tightly to prevent evaporation during cycling. A quick spin in a microcentrifuge ensures all liquid is collected at the bottom, preparing them for the thermocycler.
Programming the Thermocycler
The thermocycler must be carefully programmed to cycle through three distinct temperature steps for DNA amplification. The first step, initial denaturation, involves heating the reaction to a high temperature, 94°C to 98°C, for an extended period of one to five minutes. This step is necessary to completely separate the double-stranded template DNA into single strands and to fully activate the polymerase if a “hot-start” enzyme is used.
Following the initial step, the machine enters the main cycling phase, which is repeated 25 to 40 times. Each cycle begins with a denaturation step, where the temperature is raised again to 94°C to 98°C for about 30 seconds, causing the newly formed double-stranded DNA to separate.
Next, the temperature is rapidly lowered to the annealing stage, which lasts for 30 seconds to two minutes. The specific annealing temperature is set to be a few degrees below the melting temperature of the primers, between 50°C and 70°C, allowing the primers to bind specifically to their complementary sequences. If the temperature is too high, the primers will not bind; if too low, they may bind non-specifically, leading to unwanted products.
The final step of the cycle is extension or elongation, where the temperature is raised to the optimal working temperature for Taq polymerase, 72°C. At this temperature, the polymerase binds to the primer-template complex and begins synthesizing a new complementary DNA strand by adding dNTPs. The duration of this step is determined by the length of the target DNA, requiring one minute for every 1,000 base pairs to be copied. The thermocycler lid is also heated above 100°C to prevent water vapor from condensing on the tube walls, which would change the concentration of the reaction components.
Analyzing the Results
Once the thermal cycling program is complete, the resulting amplified DNA, known as the amplicon, is not visible to the naked eye. The success of the setup is confirmed using agarose gel electrophoresis, a standard method for separating DNA fragments. The PCR product is loaded into wells of an agarose gel, a porous matrix, and an electric current is applied.
The negatively charged DNA molecules migrate through the gel toward the positive electrode, with smaller fragments moving faster than larger ones. The gel contains a fluorescent dye that binds to the DNA, allowing the fragments to be visualized under ultraviolet light. By comparing the migration distance of the sample DNA to a molecular weight marker, or “ladder,” researchers confirm if the product is the correct size for the intended target sequence.
The presence of a distinct band at the expected size indicates a successful amplification of the target DNA. Conversely, the absence of a band or the presence of multiple, incorrectly sized bands suggests a problem with the reaction setup, such as contamination or non-specific primer binding. Analyzing the positive and negative controls run alongside the samples helps to validate the results and confirm that the reaction functioned as expected.