It is particularly useful in increasing the specificity of PCR and at times quite handy in case of multiplexing.

Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.

To be specific, generally results in specific amplification of desired PCR product without optimizing the PCR protocol.

1. Determine the melting temperature (Tm) of the primers (for the purpose of this protocol we will use 60C).

2. Set up a PCR machine with touchdown program (5 degrees higher than the Tm, that reduces ½ a degree per cycle until it reaches 5 degrees lower than the Tm).

Touchdown PCR is a technique to increase the likelyhood of amplifying your product of choice in cases where you get significant amounts of wrong priming.

In a Touchdown PCR you start with a very high annealing temperature (2-6 degrees above the one recommended for your primers) that you decrease every cycle. It is usually applied if your primers tend to bind unspecifically at temperatures close (but lower) to the annealing temperature of the correct positions. That way, the correct product gets a head start.

But if you have the capacities (and enough material), I would reccomend using a gradient with fixed annealing temperatures.

How about:

http://en.wikipedia.org/wiki/Touchdown_polymerase_chain_reaction

But if that didn't help, it is especially used when one (or both) of the primers is at risk of annealing to a non-desired site and amplifying non-specific product. By starting at the higher temperature the most specific fragment will be amplified first. Even if only one of the 2 primers works at that temperature  and you only get linear amplification rather than exponential, you do increase the amount of specific template once the annealing temperatures and will amplify that more in subsequent rounds

In simple, Touch Down PCR will have -1 deg temperature get reduce in each round of PCR amplification cycle, in which the best Annealing temp will allow your product to get amplify. with less possible for Non specific product.

It is particularly useful in increasing the specificity of PCR and at times quite handy in case of multiplexing.

Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.

To be specific, generally results in specific amplification of desired PCR product without optimizing the PCR protocol.

1. Determine the melting temperature (Tm) of the primers (for the purpose of this protocol we will use 60C).

2. Set up a PCR machine with touchdown program (5 degrees higher than the Tm, that reduces ½ a degree per cycle until it reaches 5 degrees lower than the Tm).

Thank You sir

Are touchdown and gradient PCR same ?

Krishnendu Das

Hi, it isn´t the same; in touchdown PCR you change the temperature every cycle in the same tube, and in gradient PCR you choose different temperatures for each tube searching the ideal temperature (Tm) for you reaction.