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PCR temperature: The Meaning of Optimal Over- and Undershoot
Lab Academy
- Molecular Biology
- PCR Cyclers
- Amplification & PCR
- Quality
- Reproducibility
- Accuracy
- Essay
In a PCR thermal cycler, temperature overshoot and undershoot is a necessary part of temperature cycling because it allows the whole reaction liquid in the PCR vessel to quickly reach the desired temperature. This naturally means that the critical dependent factor is the reaction volume involved.
Too high or too low temperatures will have adverse effect on the PCR. When you have too high overshoot, especially at denaturation step, you run the risk of killing off some enzyme activity (Figure 1B: Thermal cycler B with overshoot of more than 10°C). Such reduction in enzyme half-life (especially when it happens repetitively at every cycle in a PCR reaction) can decrease the total PCR yield.
Similarly, when you have a “high” undershoot (large dip in temperature), especially when applied to going down from denaturation step to annealing step, it is effectively lowering the stringency of specific primer annealing. For example, if you set annealing to 65°C, but it stays at 5°C below that temperature for many seconds due to undershoot, it can increase the chances of non-specific amplification during that period of time (Figure 1).
Similarly, when you have a “high” undershoot (large dip in temperature), especially when applied to going down from denaturation step to annealing step, it is effectively lowering the stringency of specific primer annealing. For example, if you set annealing to 65°C, but it stays at 5°C below that temperature for many seconds due to undershoot, it can increase the chances of non-specific amplification during that period of time (Figure 1).
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Figure 1: Temperature profiles of Eppendorf Mastercycler and Thermal cycler B and their respective over-shoot and undershoot algorithms.
Some thermal cycler manufacturers set high degree of over-/undershoot in their PCR temperature profile while others use sample volume prediction algorithm to change the degree of over/under-shoot required. However, this neglects the characteristic of different DNA templates. DNA templates with higher GC-content require higher energy for breaking the bonds and hence would require higher temperature at the same sample volume.
Eppendorf proprietary temperature control algorithm (Figure 2) helps ensure that the degree of overshoot and undershoot be within the optimal range of the set point temperature. This allow fast heat transfer without the associated deleterious effects.
Eppendorf proprietary temperature control algorithm (Figure 2) helps ensure that the degree of overshoot and undershoot be within the optimal range of the set point temperature. This allow fast heat transfer without the associated deleterious effects.
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Figure 2: The four temperature control modes of Mastercycler® X50 caters for different reaction volumes and/or GC-content of the DNA template and the algorithm uniquely match the suitable degree of overshoot and undershoot necessary for the entire liquid volume to reach set point temperature homogenously in the shortest time.
In other words, optimal temperature overshoot is good for fast transfer of energy from thermal block to sample in reaction vessel, leading to fast PCR completion and optimal yield and specificity. Too high overshoot or undershoot will reduce PCR yield and increase the risk of mispriming.
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