Thermal cycling design — A standard PCR cycle is composed of how many fundamental steps repeated each cycle?

Introduction / Context:
PCR relies on repeating temperature-driven steps that denature template DNA, allow primers to hybridize, and extend new strands. Recognizing the canonical step count helps when programming thermocyclers and troubleshooting amplification problems.

Given Data / Assumptions:

• We consider the classic, three-step PCR cycle.
• Some protocols merge steps (e.g., two-step PCR for high-Tm primers), but the canonical model still has three distinct phases.
• Optional initial denaturation and final extension are not counted per-cycle steps.

Concept / Approach:
The classic PCR cycle consists of: (1) denaturation (typically ~94–98°C), (2) annealing (primer binding at a temperature based on primer Tm), and (3) extension (DNA synthesis at polymerase optimum, often 68–72°C). These three are repeated for 25–40 cycles depending on the application.

Step-by-Step Solution:

Verification / Alternative check:
Manufacturer protocols for Taq polymerase describe three steps per cycle, validating the conventional model.

Why Other Options Are Wrong:

• Two steps: used in specialized protocols (e.g., long primers) but not the standard.
• Four or five steps: overcounts by splitting phases or adding non-cycle holds.

Common Pitfalls:
Confusing initial denaturation or final extension as additional per-cycle steps; they are single events outside the cycle loop.

Final Answer:
three steps