You can also set the final annealing temperature 1–2☌ below the calculated Tm. The cycles and temperature drop during the touchdown phase can be adjusted from 1–3 cycles per 1-3☌ drop in temperature if non-specific products are still observed or if the yield is low. Table 1: An example of a touchdown PCR protocol based on a Tm of 57☌. Phase 2 follows generic PCR amplification of up to 20–25 cycles using the final annealing temperature reached in the touchdown phase (Table 1). This is done for a total of 10–15 cycles. The temperature is reduced by 1☌ every successive cycle until the calculated Tm range is reached. The first phase of touchdown programming uses a Tm that is approximately 10☌ above the calculated Tm. The suggested cycling program has two phases. There is a protocol published in Nature Protocols, which works very well and is a great reference to start off with for touchdown PCR. The stepwise transition to a lower temperature during the subsequent cycles guards against lower yields by making use of the desired amplicons in the reaction that now outcompete any non-specific products or primer-dimers if present. Too low annealing temperatures can lead to primer-dimer formation and non-specific products while too high temperatures can reduce yield due to poor primer annealing.īy using temperatures higher than the calculated Tm in the initial cycles, touchdown PCR favors the accumulation of amplicons whose primer-template complementarity is the highest. Therefore, it is often difficult to find the right annealing temperature for a given primer/template combination. This is normally determined based on the Tm of the primer-template pair.īut, primer Tm can be affected by the individual buffer components, and even primer and template concentrations, meaning any calculated primer Tm value is only an approximation. In all types of PCR, it is essential to determine the optimal annealing temperature. Image credit: Laura Grassie.Ī gradual lowering of temperature to a more permissive annealing temperature during the course of cycling favors amplification of the desired amplicon. The initial annealing temperature is set higher than the optimal melting temperature (Tm) of the primers and is then gradually reduced over subsequent cycles until the Tm or “touchdown temperature” is reached, much like the touchdown of an airplane onto the runway (Figure 1).įigure 1: Representation of touchdown PCR cycles and how annealing temperature changes. Touchdown PCR is a modification of PCR used to help reduce non-specific annealing and amplification. Optimizing the PCR conditions by testing various annealing temperatures and other conditions is another way, although this can be time-consuming to complete.Īnother, less time- and resource-intensive option, is to use touchdown PCR.
Touchdown pcr vs gradient pcr how to#
How to Eliminate Non-Specific Amplification in PCRĬareful design of PCR primers can help reduce the change of off-target binding, although the sequence of the desired target DNA does limit the choice. Mispriming, where primers bind non-specific sequences in the DNA template, resulting in the DNA polymerase amplifying the wrong template DNA sequence, is one cause of non-specific bands on gel following PCR. So what’s the issue with non-specific amplification? PCRs can, and often do, suffer from issues such as non-specific amplification. The Problem of Non-Specific Amplification So in this article, I’ll provide a primer on touchdown PCR and give you 5 key tips and references for perfecting it. However, using this technique in the lab can be trickier than it might seem at first. Touchdown PCR is a very useful technique for improving PCR amplification specificity and sensitivity.
To be honest, 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. There are many different types of polymerase chain reaction (PCR) you might have heard of or want to use for a specific requirement in the lab.
0 kommentar(er)
