All of the following statements regarding the polymerase chain reaction (PCR) are correct EXCEPT:

Explanation

## PCR Cycle Phases and Primer Annealing **Key Point:** The annealing temperature in PCR is typically set **3–5°C BELOW** the melting temperature (Tm) of the primers, not above. Setting it above Tm would prevent primer annealing altogether, resulting in no amplification. ### Correct PCR Principles | Phase | Temperature | Duration | Purpose | |-------|-------------|----------|----------| | Denaturation | 94–95°C | 15–30 sec | Separate double-stranded DNA into single strands | | Annealing | Tm − 3 to 5°C | 20–30 sec | Allow primers to bind to complementary sequences | | Extension | 72°C | 1–2 min/kb | Taq polymerase synthesizes new DNA strand | **High-Yield:** The three correct statements are: 1. **Taq polymerase** — isolated from *Thermus aquaticus*, remains active at >90°C; this is why it is the enzyme of choice for PCR. 2. **Exponential amplification** — each cycle doubles the target, yielding 2^n copies after n cycles (e.g., 30 cycles = ~10^9 fold amplification). 3. **Extension at 72°C** — optimal temperature for Taq polymerase activity; extension time depends on amplicon length (~1 min per kilobase). **Mnemonic: DAE** — **D**enaturation (95°C), **A**nnealing (Tm − 5°C), **E**xtension (72°C). **Clinical Pearl:** Incorrect annealing temperature is a common cause of PCR failure in the laboratory. Too high → no primer binding; too low → non-specific amplification and primer dimers.