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Table 2 Melting temperature (Tm°C) and annealing temperature (Ta°C) data obtained from fluorometric melting curve analysis

From: Enhanced annealing of mismatched oligonucleotides using a novel melting curve assay allows efficient in vitro discrimination and restriction of a single nucleotide polymorphism

Mismatch Set Observed Tm Predicted Tma ΔTm (Obs -Pred) ΔTm -ΔTm(Ave)b Optimal Ta ΔTa (Obs Tm - Ta) Corrected Ta
wt/wt 78 67 11 0.7 60 18 49.7
wt/mut 71 61.8 9.2 -1.1 50 21 39.7
wt/1 × 1 69 60.2 8.8 -1.5 50 19 39.7
wt/2 × 1 66 56.1 9.9 -0.4 50 16 39.7
mut/mut 79 68.7 10.3 0 60 19 49.7
mut/wt 74 63.1 10.9 0.6 50 24 39.7
mut/1 × 1 66 56 10 -0.3 50 16 39.7
mut/2 × 1 64 51.7 12.3 2 50 14 39.7
Average 5.0 ± 2.28 SDc 5.33 ± 1.09 SDc ΔTm(Ave) = 10.3d    18.38 ± 3.16 SDe  
  1. a Predicted Tm was determined using an online Tm calculator (http://unicorn.ps.uci.edu/calculations/DNA/DNAthermocalc.html), which initially calculates a Tm value as if the mismatch was not present, and then subsequently corrects this initial value by the addition of a destabilisation factor dependant on the identity of the mismatch being either a dG, dC, or neither a dG/dC [23].
  2. b ΔTm-ΔTm(Ave) is used as a relative measure of observed annealed complex stability relative to predicted values. A value of zero indicates there is no deviation between observed and predicted Tm values. Annealed templates are considered less-stable as the value becomes more negative, whereas a more positive value indicates greater template stability than predicted.
  3. c Values indicate mean temperature change per single base increase/decrease in "bubble" size.
  4. d Average ΔTm describes the temperature difference as a result of the LC Green dye binding to dsDNA. This value is used to correct the annealing temperature.
  5. e Average of difference in observed Tm and observed annealing temperature, indicative of the decrease away from the Tm needed to achieve optimal annealing conditions.