Negative Amplification Control
Definition
A no-template reaction well run alongside case samples; any amplified peak indicates reagent contamination or analyst-introduced contamination during setup.
- Field
- Forensic DNA, PCR quality control
- Contents
- No-template reaction well
- Run alongside
- Case samples in the same batch
- Positive result meaning
- Reagent or setup contamination
Common questions
What does an examiner do if the negative amplification control shows a peak?+
A positive result in the no-template control undermines confidence in the whole batch, since it means contamination entered somewhere in the reagents or handling, so the batch is typically investigated for the contamination source and reprocessed rather than the case results being reported as if nothing occurred.
How is a negative amplification control different from a negative extraction control?+
An extraction control tests for contamination introduced during DNA extraction from a blank sample carried through the whole process, while the amplification control isolates just the PCR step by using no template at all, which helps narrow down at which stage of the workflow any contamination entered.
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...
- Extension
- The PCR step at ~72°C at which thermostable DNA polymerase synthesises a new complementary strand from each primer, using dNTPs in the...
- 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.
- 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...