Quantcast
Skip to content

Touchdown PCR: A Primer and Some Tips

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:

  1. Keeping all reactions cold until thermal cycling starts is crucial to avoiding non-specific priming even with TD-PCR.
  2. 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.
  3. TD-PCR can address problems with monoplex reactions better than multiplex reactions.
  4. 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.
  5. An extra 1 min denaturation cycle at 96°C or 97°C may be extremely useful for difficult templates.

For more information on protocol and optimization, see references 1 and 2. I also found the Roux KH paper very useful for optimization in general.

References

  1. Roux KH. Genome Research. 1995. 4: S185-194.
  2. Korbie DJ and Mattick JS. Nature Protocols. 2008. 3(9). 1452 – 1456.

Originally published on Jul 20, 2009. Updated and Revised on August 6, 2015.

10 Comments

  1. Emma Farquharson on December 21, 2016 at 9:23 pm

    This was an excellent post. It was clear, had references, and pointed out a pretty clear protocol. Thank you for the help!

  2. Jessica on February 22, 2011 at 2:44 am

    Hi, Shoba,

    It’s nice to get some useful information from you,would you please recommend a nice PCR instrument to do Toughdown PCR? I am going to buy a new one.How about ABI GENEaMP pcr system 9700? Is it good? Or too old?

    Thanks a lot.

  3. ms maryem on March 18, 2010 at 8:35 pm

    well, this is not a comment i want more information regarding each of the terms in the above article, so that i can well understand PCR and every thing related to it.
    Thank you

    Ms. maryem
    Qc. Canada

    • Shoba on March 19, 2010 at 2:33 pm

      Hi Maryem,

      Information on basic PCR can be found in any introductory molecular biology textbook or book on molecular biology techniques.

  4. John Mackay on July 21, 2009 at 8:49 pm

    Indeed Kurt – hence my Australia comment. Since I am not a fan of acronyms, I think we can also note that this technique was published without one. It may have been the last adaption of PCR to have been so 😉

  5. Kurt on July 21, 2009 at 5:00 am

    I THINK the original Touchdown PCR reference should be:

    Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ‘Touchdown’ PCR to
    circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991
    Jul 25;19(14):4008. PubMed PMID: 1861999

  6. John Mackay on July 20, 2009 at 9:11 pm

    Also very useful if you’re running several different reactions in the same plate – allows you to accomodate multiple primer Tm’s. Like Jon its a case of ” Hi, I’m John and I’d like you to know I’ve been a big fan of your’s for a long time”.
    One of the best things to come out of Australia ! (from a NZ’er)

  7. Cameron Gundry on July 20, 2009 at 6:35 pm

    Hello from Idaho Technology:

    I’ve done somewhere between 5,000 and 20,000 PCR experiments runs for more than 13 years (the geek-factor is high, but I’m proud of it) and the reason that TD-PCR is good for the end result is that it eliminates those pesky undesirable products like Shoba mentions.

    However, I’d like to ad that PCR problems are often a result of poor primer design. Alghouth TD-PCR can often help you amplify pretty clean stuff despite sub-optimal primer design, it’s not magic and it’s still better to start w/ good primers from the beginning. And, it doesn’t have to be painstaking. The time has passed to just pick primers out of the blue and optimize for days or weeks untill it works. Costly mistakes can be avoided by avoiding the “bad things” and beginning with good primer design.

    Bad thing #1) primer homologies with duplicated genes and so on.
    Bad thing #2) self-complimentarities and other primer problems

    Here’s where I get long-winded. It’s also interesting to think that the efficiency of the PCR would be changing a fair amount during all those 10-15 cycles of lowering the annealing temperature. I think that the efficiency would be increasing dramatically once the annealing temp is low enough for the primers to actually begin annealing. I’ve wondered in the past if this matters. So, does TD-PCR limit you? I guess that so long as you don’t need a really wide dynamic range of detection it might not matter because once the qPCR machine begins registering signal growth, it’s probably a few cycles later than all that “touching down” anyways. Besides, in any PCR the eficiency probably always in some flux, in general, gradually increasing before plateauing and reaching zero after exhausting the resources in the master mix. Such is the inhale and exhale of life in a PCR reaction.

  8. Jon on July 20, 2009 at 2:27 pm

    I’ve been a Long-time follower of touch-down PCR, especially for my colony PCRs.

    My favourite programme actually consists of decreasing the Tm from 65 to 55oC by 1oC every cycle for 10 cycles, and subsequently continuing the reaction for another 25 cycles at 55oC. I have been able to use some very non-optimal primers to amplify very large colony PCR bands (>40bp primers amplifying >4kb bands).

    Sometimes I also think that the 65/55oC TD protocol is the ‘holy grail’ of PCR protocols, as most of my PCRs require little tweaking around those settings.

  9. Greta on July 20, 2009 at 12:20 pm

    Hi,
    I have used TD-PCR many times and has often solved difficult situations!

Leave a Comment





This site uses Akismet to reduce spam. Learn how your comment data is processed.

Scroll To Top
Share via
Copy link