In a standard three-step PCR cycle, what is the correct sequence of temperatures typically used?

• A Denaturation at 94 to 95 degrees C, primer annealing at 50 to 60 degrees C, extension at 72 degrees C ✓
• B Annealing at 94 to 95 degrees C, denaturation at 55 degrees C, extension at 72 degrees C
• C Extension at 94 to 95 degrees C, denaturation at 72 degrees C, annealing at 55 degrees C
• D Denaturation at 72 degrees C, annealing at 94 to 95 degrees C, extension at 55 degrees C

Explanation

Each cycle begins with denaturation of the template at about 94 to 95 degrees C, followed by primer annealing at roughly 50 to 60 degrees C depending on primer melting temperature, then extension at 72 degrees C, the optimum for Taq DNA polymerase. Option B reverses the first two steps, which would prevent strand separation before primers bind. Extension never occurs at denaturation temperature because the enzyme would be stable but primers would not remain annealed.

Reference: Lippincott's Illustrated Reviews: Biochemistry, 8th ed.

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