The Polymerase Chain Reaction, or PCR, is a laboratory method used to rapidly create millions or even billions of copies of a specific segment of DNA. It allows for the generation of large quantities of DNA from a very small initial amount. This powerful process underpins many procedures in genetic testing, research, and the identification of infectious agents.
Key Chemical Ingredients
For PCR to occur, several specific chemical ingredients must be present in the reaction mixture. Each component plays a distinct and important role in the amplification process, working together to synthesize new DNA strands.
The DNA template is the initial DNA sample containing the specific sequence to be copied. This target DNA serves as the pattern for new strands. Even a minute amount of template DNA can be sufficient for amplification.
Primers are short, synthetic single-stranded DNA fragments. Two primers are used in each PCR reaction: a forward primer and a reverse primer. These primers are designed to bind to opposite ends of the target DNA sequence, defining the specific region that will be amplified. They serve as a starting point for DNA synthesis because DNA polymerase cannot initiate new strands on its own.
DNA polymerase is an enzyme that synthesizes new DNA strands by adding nucleotides. For PCR, a special type of thermostable DNA polymerase is used, most commonly Taq polymerase. This enzyme is isolated from the bacterium Thermus aquaticus, allowing it to withstand the high temperatures required during the PCR process without denaturing. This thermostability is important because the mixture is repeatedly heated to separate DNA strands.
Deoxynucleotide triphosphates, or dNTPs, are the individual building blocks (adenine, guanine, cytosine, and thymine) that the DNA polymerase uses to construct new DNA strands. These molecules provide the necessary nucleotides to extend the growing DNA strand, binding to the complementary DNA template. They are supplied in equal molar concentrations.
A reaction buffer provides the optimal chemical environment for the DNA polymerase to function effectively. This includes maintaining a stable pH and containing salts like potassium chloride (KCl) and magnesium chloride (MgCl2). Magnesium ions are important cofactors for DNA polymerase activity and promote primer annealing.
Essential Equipment
PCR relies on specialized equipment that precisely controls the reaction conditions. While basic laboratory tools are needed for preparation, one machine is central to the process.
The thermocycler, also known as a PCR machine, is the primary piece of equipment. This instrument automates the process of rapidly and precisely changing the temperature of the reaction tubes. It cycles through specific temperatures for defined durations. The development of the thermocycler, alongside the discovery of Taq DNA polymerase, made PCR automation a reality, replacing the laborious manual transfer of tubes between water baths.
Beyond the thermocycler, other standard laboratory equipment is necessary for preparing the reactions. This includes sterile tubes, pipettes for accurate measurement and transfer of liquids, and pipette tips to prevent contamination.
The Amplification Cycle
The amplification cycle describes how the chemical ingredients and the thermocycler work together to exponentially increase the number of target DNA copies. This cyclical process involves three main temperature-dependent steps, repeated multiple times.
The first step is denaturation, where the reaction mixture is heated to a high temperature. This heat breaks the hydrogen bonds between the complementary bases of the double-stranded DNA template, separating it into two single strands. This separation is important, as the single strands will serve as templates for new DNA synthesis.
Next is annealing, where the temperature is lowered to allow the primers to bind to their specific sequences on the single-stranded DNA templates. The annealing temperature usually ranges from 50-65°C, and its precise setting is important for specific primer binding. During this phase, the forward and reverse primers attach to their complementary sites at the ends of the target DNA region.
The final step in each cycle is extension, where the temperature is raised again. This temperature is optimal for the Taq DNA polymerase enzyme, allowing it to synthesize new DNA strands. Starting from the annealed primers, the polymerase adds dNTPs to extend the new strand complementary to the template. This process results in two new double-stranded DNA molecules for every original template.
These three steps—denaturation, annealing, and extension—are repeated for multiple cycles, typically 20-40 times. Each cycle theoretically doubles the amount of the target DNA, leading to an exponential increase in copies. For instance, 20 cycles can produce over a million copies, and 30 cycles can yield over a billion.