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 | |
- 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].
- 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.
- c Values indicate mean temperature change per single base increase/decrease in "bubble" size.
- 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.
- 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.
