The Polymerase Chain Reaction (PCR) is a laboratory technique used to create millions of copies of a specific segment of DNA very rapidly. This process mimics natural DNA replication but occurs in a test tube, providing researchers with a large sample for study, identification, or diagnosis. PCR is a foundational tool in molecular biology, enabling applications from forensic analysis and genetic research to the detection of infectious agents. The method works by subjecting a reaction mixture containing the target DNA to repeated cycles of heating and cooling.
The Essential Ingredients
A successful PCR requires five main components combined in a small reaction tube.
DNA Template
This is the specific sequence of genetic material intended for amplification. The template can originate from various sources, such as a patient sample or a biological specimen.
Primers
These are short, synthetic strands of DNA that provide a starting signal. A pair of primers is designed to be complementary to the sequences flanking the target DNA region, marking the precise start and end points for the desired product.
Taq Polymerase
This specialized enzyme builds the new DNA strands. It is a heat-stable DNA polymerase isolated from the bacterium Thermus aquaticus, allowing it to withstand the high temperatures required during cycling without degrading.
Deoxynucleotide Triphosphates (dNTPs)
These are the four bases (adenine, guanine, cytosine, and thymine) that serve as the raw material for synthesizing new DNA strands. The Taq polymerase links these building blocks together.
Buffer Solution
The buffer maintains the optimal chemical environment for the Taq polymerase to function. It often contains magnesium ions ($\text{Mg}^{2+}$), which act as a necessary cofactor to activate the enzyme and stabilize the interaction between the primers and the template DNA.
The Cyclic Mechanism of Amplification
Once all components are combined, the reaction proceeds through a series of temperature shifts that form the core mechanism of PCR.
Denaturation
The mixture is heated to a high temperature, typically between $94^{\circ}\text{C}$ and $98^{\circ}\text{C}$, for $20$ to $30$ seconds. This intense heat breaks the hydrogen bonds holding the double-stranded DNA template together, separating them into two single strands. This separation creates the necessary templates for new copies.
Annealing
The temperature is rapidly lowered, usually to $50^{\circ}\text{C}$ to $65^{\circ}\text{C}$. This cooler temperature allows the synthetic primers to bind (anneal) specifically to their complementary sequences on the single-stranded DNA templates. The annealing temperature is critical for ensuring the primers attach only to the intended target region.
Extension
The temperature is raised slightly to approximately $72^{\circ}\text{C}$, the optimal working temperature for Taq polymerase. Starting from the $3^{\prime}$ end of each bound primer, the Taq polymerase synthesizes a new complementary DNA strand. The enzyme incorporates free dNTPs, pairing them with the exposed bases on the template strand to create a complete, double-stranded DNA molecule. At the completion of this step, the total amount of the target DNA sequence has doubled.
Scaling and Automation
The three-step temperature cycle is repeated multiple times to achieve significant DNA amplification. This cyclic process is managed by a specialized laboratory instrument called a thermal cycler, which is an automated heating and cooling block. The thermal cycler precisely controls the temperature changes and duration required for each step.
A typical PCR program repeats this cycle between $25$ and $40$ times, driving an exponential increase in the target DNA. Since the amount of DNA product doubles with every cycle, amplification follows the formula $2^n$, where $n$ is the number of cycles. Starting with a single molecule of target DNA, $30$ cycles can produce over a billion copies, making the technique powerful for detecting minute quantities of genetic material.
Interpreting the Outcome
After the programmed number of cycles, the reaction tube contains billions of copies of the target DNA (the amplicon), but this product is too small to be seen directly. Researchers must employ a visualization technique to confirm the reaction was successful and that the correct DNA segment was amplified.
The most common method for this analysis is gel electrophoresis, which separates DNA fragments based on size. The DNA products are loaded into wells in a slab of agarose gel, and an electric current is applied. Because DNA molecules carry a negative charge, they migrate through the gel matrix toward the positive electrode. Smaller DNA fragments move more easily and travel farther through the porous gel than larger fragments.
To visualize the separated DNA, the gel is treated with a fluorescent dye that binds to the DNA strands. This allows the fragments to be seen as distinct bands under ultraviolet light. The presence of a band at the expected length, confirmed by comparing it to a standard ladder of known sizes, verifies the successful amplification of the target sequence for further analysis.