A-Addition
Definition
The non-templated addition of a single adenine nucleotide to the 3' end of PCR products by Taq polymerase, exploited in forensic STR kits via a 60-minute final extension to drive all alleles to the +A form for consistent CE sizing.
- Mechanism
- Non-templated adenine added to 3' end
- Enzyme responsible
- Taq polymerase
- Forensic fix
- Extended final extension step (~60 min)
- Field
- STR typing via PCR
Common questions
Why does incomplete A-addition cause a problem in STR results?+
If only some copies of an allele gain the extra adenine, the fragment population splits between the +A and non-A lengths, producing a split or shouldered peak on the electropherogram instead of one clean peak, which complicates sizing and interpretation.
How do STR kits prevent this from happening?+
Kits build in a long final extension step, commonly around 60 minutes, that gives Taq polymerase enough time to add the extra adenine to essentially every copy, driving the whole population to the consistent +A form.
Related terms
- 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.
- 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...