Step by Step
D
Step 1: Denaturation — 95°C
Heat the reaction to ~95°C. The hydrogen bonds between the two DNA strands break apart — the double helix separates into two single strands. These will serve as templates.
Memory trick: D = "Divide" — the strands split apart like a zipper opening.
A
Step 2: Annealing — ~55°C
Cool the reaction to ~55°C. Short DNA sequences called primers bind (anneal) to their complementary sequences on each template strand. Primers define the region to be amplified and provide a starting point for synthesis.
Memory trick: A = "Attach" — primers snap onto their target sequences.
E
Step 3: Extension — 72°C
Heat to 72°C — the optimal temperature for Taq polymerase (a heat-stable DNA polymerase from Thermus aquaticus). Taq extends from the primers, synthesizing a new complementary strand 5'→3'. Each cycle doubles the amount of target DNA.
Memory trick: E = "Extend" — Taq polymerase builds the new strand.
2ⁿ
Exponential amplification
Each cycle doubles the DNA. After 30 cycles: 2³⁰ ≈ 1 billion copies. PCR can amplify a single DNA molecule into enough material for sequencing, forensic analysis, or diagnostic testing (e.g., COVID-19 PCR tests).
Applied Walkthrough
1
A crime scene yields a tiny blood sample. Forensic scientists need to analyze the DNA but there isn't enough for testing.
2
PCR is used: the target region is heated to 95°C (denaturation), cooled to 55°C (primers anneal), then 72°C (Taq extends).
3
After 30 cycles, the original trace DNA has been amplified to ~1 billion copies — enough for gel electrophoresis and identification.
4
The same principle applies to COVID-19 PCR tests — detecting viral RNA (converted to DNA first by reverse transcriptase) and amplifying it until detectable.
Exam Application
Exams test the three steps in order (denaturation, annealing, extension), the temperatures for each, the role of Taq polymerase, and why Taq specifically is used (heat-stable — survives 95°C denaturation). Also know that PCR requires primers — it cannot amplify DNA without them.
⚠ Common Trap
Students confuse the order of temperatures or forget why Taq polymerase is used instead of regular DNA polymerase. Regular polymerase would denature at 95°C — Taq survives it because it comes from bacteria that live in hot springs. Also: PCR amplifies a specific region defined by the primers — it does NOT sequence the DNA by itself.
✓ Quick Self-Check
1. What are the three steps of PCR in order?
Denaturation (95°C), Annealing (~55°C), Extension (72°C).
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2. Why is Taq polymerase used in PCR instead of regular DNA polymerase?
Taq polymerase is heat-stable — it survives the 95°C denaturation step. Regular polymerase would be destroyed.
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3. What is the role of primers in PCR?
Primers define the region to be amplified and provide a starting point for Taq polymerase — DNA synthesis can only extend from a primer.
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4. How many copies of DNA are produced after 30 PCR cycles?
2³⁰ ≈ 1 billion copies.
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5. What happens during the annealing step?
The reaction is cooled to ~55°C and primers bind (anneal) to their complementary sequences on the single-stranded DNA templates.
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