Melting Temperature (Tm)
Definition
The temperature at which half of a primer-template duplex population is dissociated. Depends on primer length, GC content, and nearest-neighbour stacking interactions.
- Defined as
- Temperature where 50% of duplex is dissociated
- Depends on
- Primer length, GC content, stacking
- Used in
- PCR primer and probe design
Common questions
Why do PCR protocols specify an annealing temperature below Tm rather than at Tm?+
At Tm only half the primer is bound to template, which gives weak, inconsistent amplification. Annealing temperatures are typically set a few degrees below the calculated Tm so binding is strong and specific across the whole reaction.
Why can two primers with the same length give different Tm values?+
Tm is not just a function of length. GC content raises Tm because G-C base pairs have three hydrogen bonds versus two for A-T, and the specific sequence of neighbouring bases changes stacking energy, so nearest-neighbour models are used for accurate prediction rather than simple length-based rules.
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.
- 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...