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When I first heard of touchdown PCR, I thought of a landing aircraft, which, as it turns out is not a bad way to think about it.
But despite it’s amenability to analogies and dreadful puns (see title), touch-down PCR (TD-PCR), a very useful technique for improving PCR amplification specificity, is trickier that it might seem at first. So in this article I’ll provide a primer on touchdown PCR (TD-PCR) and some tips and references for perfecting it.
So let’s start with…
What is touchdown PCR?
TD-PCR is a modification of PCR in which the initial annealing temperature is higher than the optimal Tm of the primers and is gradually reduced over subsequent cycles until the Tm temperature or “touchdown temperature” is reached, much like the touchdown of an airplane.
A gradual lowering of temperature to a more permissive annealing temperature during the course of cycling favors amplification of the desired amplicon.
Why does TD-PCR work better?
Optimal annealing temperature is a requirement in PCR. This is normally determined based on the melting temperature (Tm) of the primer-template pair. But, primer Tm is affected variously by the individual buffer components, even primer and template concentrations so any calculated primer Tm value is only an approximation (1). Therefore, it is often difficult to find the right annealing temperature for a given primer/template combination.
To-low annealing temperatures, can lead to primer-dimer formation and non-specific products while too-high temperatures reduce yield due to poor primer annealing.
By using temperatures higher than the calculated Tm in the initial cycles, TD-PCR favours only accumulation of amplicons whose primer-template complementarity is the highest. The stepwise transition to a lower temperature during subsequent cycle guards against lower yields by making use of the desired amplicons in the reaction that now outcompetes any non-specific products or primer-dimers if present.
What are TD-PCR cycling conditions?
The protocol published in Nature Protocols (2) works very well and is a good reference to start off with TD-PCR.
The suggested cycling program has two phases. The first phase of touchdown programming uses a Tm that is approximately 10°C above the calculated Tm. The temperature is reduced by 1°C every successive cycle until the calculated Tm range is reached. This is done for a total of 10-15 cycles.
Phase 2 follows generic PCR amplification of up to 20-25 cycles using the final annealing temperature reached in the touchdown phase.
The cycles and temperature drop during touchdown phase can be adjusted from 1-3 cycles per 1-3°C drop in temperature if non-specific products are still observed or if the yield is low.
General Tips for TD-PCR:
- Keeping all reactions cold until thermal cycling starts is crucial to avoiding non-specific priming even with TD-PCR.
- A hot start setup is preferred. Since the main aim of TD-PCR is to eliminate non-specific interactions during the initial cycles, it is important to use a hot-start set up.
- TD-PCR can address problems with monoplex reactions better than multiplex reactions.
- Total number of PCR cycles, including the touchdown phase should be kept low (below 35). Too many cycles will lead to appearance of non-specific bands in the gel.
- An extra 1 min denaturation cycle at 96°C or 97°C may be extremely useful for difficult templates.
- Roux KH. Genome Research. 1995. 4: S185-194.
- Korbie DJ and Mattick JS. Nature Protocols. 2008. 3(9). 1452 – 1456.