Extension
Definition
The PCR step at ~72°C at which thermostable DNA polymerase synthesises a new complementary strand from each primer, using dNTPs in the reaction buffer.
- PCR step
- Extension
- Approx. temperature
- ~72C
- Enzyme
- Thermostable DNA polymerase, e.g. Taq
- Substrate used
- dNTPs from the reaction buffer
Common questions
Why is roughly 72C used for the extension step?+
It is close to the optimal working temperature for thermostable polymerases such as Taq, at which the enzyme synthesises new DNA strand efficiently while remaining stable across the many heating and cooling cycles of a PCR run.
What happens if extension time is too short for the target amplicon length?+
The polymerase may not complete synthesis of the full-length product before the cycle moves to denaturation, producing truncated or incomplete amplicons and reducing the yield of the correctly sized target.
Related terms
- A-Addition
- The non-templated addition of a single adenine nucleotide to the 3' end of PCR products by Taq polymerase, exploited in forensic STR...
- AmpliTaq Gold
- A chemically modified hot-start form of Taq polymerase (Applied Biosystems) that is inactive at room temperature and requires a 10-11 minute activation...
- Annealing Temperature
- The reduced temperature (50-65°C) at which primers hybridise to single-stranded template. Determined by primer Tm; set to balance specificity against yield.
- Denaturation
- The separation of double-stranded DNA into two single strands by disrupting the hydrogen bonds between base pairs. In PCR, denaturation is achieved...
- Hot-Start Polymerase
- Any Taq formulation (antibody-mediated, chemically modified, or aptamer-blocked) that is inactive below ~60°C, preventing non-specific amplification during reaction setup.
- Melting Temperature (Tm)
- The temperature at which half of a primer-template duplex population is dissociated. Depends on primer length, GC content, and nearest-neighbour stacking interactions.
- Negative Amplification Control
- A no-template reaction well run alongside case samples; any amplified peak indicates reagent contamination or analyst-introduced contamination during setup.
- Uni-Directional Workflow
- The physical principle governing forensic DNA laboratory layout: personnel and materials move from pre-PCR to post-PCR zones only, preventing high-copy-number amplified products...