Touchdown and Stepdown PCR

Touchdown PCR

Touchdown PCR is a very clever and commonly used method of PCR aimed at increasing PCR specificity while still achieving a good yield of the target amplicon.

In standard PCR, the annealing temperature step is usually static and the optimal temperature is a compromise of specificity and yield, usually 3-5 °C below the calculated melting point (T_m) of the primers.

However, in touchdown PCR the PCR starts off with an annealing temperature that is higher than is optimal for the primers.

The PCR program then incrementally decreases the annealing temperature each cycle until it is below the optimal temperature for the primers. This is often done over around 10 cycles in small steps of around 1 °C. The remainder of the PCR then continues with the lower annealing temperature.

For example, if you would normally use an annealing temperature of 54 °C, a touchdown PCR may start with an annealing temperature of 60 °C and drop down 1 °C /cycle over 10 cycles, and then continue at 50 °C for a further 30 cycles.

In the early PCR cycles this process gives extremely high specificity but low yield. The advantage here is that the PCR is being slowly enriched only with the target DNA amplicons, and with very minimal non-specific amplification.

When the PCR reaches the optimal annealing temperature and then drops below it, the efficiency and yield of amplification become much higher, but the specificity is now lower than in the early cycles. However, at this point the lower specificity doesn’t matter because the majority of DNA present is already the target amplicon.

Overall, this results in a high yield of very specifically amplified DNA fragments, and a much lower chance of producing non-specific amplification compared to standard PCR using a single annealing temperature.

Stepdown PCR

Step-down PCR is a simplification of touch-down PCR that is useful for older or basic thermocyclers that lack touch-down functionality. Instead of a gradual decrease in annealing temperature, the decrease occurs in sharper steps that are easier to individually program into these devices.

For example, a program could have three cycles with annealing at 62 °C, three cycles at 58 °C, three cycles at 54 °C, and 29 cycles at 50 °C. The more steps, the more specific the PCR, but even two steps can make a big difference.

Additional applications

Touchdown and stepdown PCR are also very useful for PCRs where you are not sure what the optimal annealing temperature should be, either because you are using the primers on a range of different organisms that may have some mismatches in their primer binding sites, or because you have modified the PCR in some way without a lot of optimisation (e.g. added MgCl₂). 

Because both methods gradually drop the annealing temperature through a range of temperatures, it is not essential to know exactly what the best annealing temperature is for that application – they “find” it automatically to maximise both specificity and yield.

Further reading and resources

A really nice early illustration of touch-down and step-down PCR can be found in the following article:

Hecker, K. H., & Roux, K. H. (1996). High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques, 20(3), 478-485.

You can also learn how to program Bento Lab Pro for touchdown PCR in our online manual.

For those with Bento Lab Entry, which lacks touchdown capability, you will need to program a stepdown PCR instead. It is a little bit more difficult to program but should give you a similar result for most applications.