PCR is a laboratory technique that creates millions of copies of a specific DNA segment from a small initial sample. This process cycles through three phases: denaturation, annealing, and extension. The extension phase is the third step in each cycle, where new DNA strands are synthesized. This synthesis effectively doubles the amount of target DNA present with every cycle.
Essential Components for DNA Synthesis
The extension step requires specific molecular components to build the new DNA strand. The primary actor is a thermostable enzyme, most commonly Taq polymerase. Taq polymerase is isolated from a heat-tolerant bacterium, allowing it to remain active after exposure to the high temperatures of the denaturation step.
The polymerase requires short, single-stranded DNA primers to begin synthesis. These primers bind to the template DNA during the annealing phase, providing a starting point. The final necessary ingredients are deoxynucleotide triphosphates (dNTPs), which serve as the building blocks for the new strand. dNTPs include all four bases—adenine, guanine, cytosine, and thymine—ready to be incorporated into the growing chain.
The Mechanism of Extension
The extension phase begins when Taq polymerase binds to the double-stranded region formed by the primer. The enzyme positions itself at the primer’s 3′ end, which initiates the lengthening of the strand. The polymerase then reads the sequence of bases on the single-stranded template.
As the enzyme moves, it recruits complementary dNTPs from the solution. For instance, if the template has an adenine (A), the polymerase incorporates a thymine (T) into the new strand. The polymerase forms a phosphodiester bond to link the added dNTP to the growing chain. This process is directional, meaning the new DNA strand is always synthesized in the 5′ to 3′ direction.
Synthesis continues until the polymerase reaches the end of the template strand or the region defined by the second primer. The single-stranded template DNA is converted into a new double-stranded DNA molecule. Since both original template strands are copied, the amount of target DNA is doubled in a single cycle.
Why Timing and Temperature Matter
The efficiency and accuracy of the extension step depend on precise temperature control. A temperature of approximately 72°C is used because it is the optimal operating temperature for Taq polymerase. This temperature ensures the enzyme achieves maximum polymerization speed and stability.
The duration of the extension step relates directly to the length of the DNA segment being amplified. A general rule for standard Taq polymerase is to allow one minute of extension time for every 1,000 base pairs (one kilobase or kb). Longer DNA fragments require a longer extension period to ensure the polymerase fully completes the new strand. Once the time is complete, the temperature rapidly increases to begin the next denaturation step, allowing the newly synthesized strands to serve as templates in subsequent cycles.