This would require 7 cycles and 14 cycles respectivelyģ. You can drop the temp down in the example cited by 6C 0.5C/cycle or 1C/cycle. to 57C (in reactions where Tms differ go for Tm + 3 of the HIGHEST Tm primer) to Tm-3C thus 57C in the example citedĢ. denaturation annealing and extension but in the middle cycle you drop the annealing temp in the same reaction from Tm + 3C thus for example 63C where the Tms of primers approx. In the initial phase of TD you have a 3 cycle set up, i.e. In essence, there are 2 3 step cycles to touchdown PCR unlike standard PCR with or without differing annealing temp in different physical reactions, i.e. See the bottom for a typical touch down set up and ref paper supplied Where your forward and reverse primer Tms differ by more than 3C Where standard PCR has been performed and you have multiple non specific productsĬ. In essence, touch down tends to be used if you requireī. Touch down PCR will perform the same function but is slightly more specialised and requires a PCR block where you can drop the annealing temp cycle to cycle WITHIN THE SAME TUBE Thus your optimal PCR going forward would be a standard 3 step PCR but with a single (empirically defined optimal) Ta of 58C The Object of this exercise is to find the optimal PCR in terms of a single specific band made with high efficiency: Thus, at Tm-5C (55C) you might find a lot of your specific product but small traces of a non specific product (but not always) at Tm-2C (58C) you might get the same amount of specific product but not your non specific product and at Tm (60C) you might see no non specific product but lower yields of your specific amplicon See gradient PCR as an example of how this reaction would be configured immediately below: Apart from the Ta everything else in the 6 sets of reactions is identical and the same variant annealing temp is used through out the whole PCR Typically 30-35 cycles (see below) So for example if your predicted primer Tm was 57C, then you would set up 6 separate sets of PCR reactions where the annealing temp in the first set was 55C in the second set 56C in the third set 57C and finally in the 4th row of the PCR block 58C in the 5th 59C and finally in the 6th row a Ta of 60C Typically, you vary the annealing temp across the block by 1C per row starting Tm-5C and going up to the TM However, with Gradient PCR you perform a standard 3 step PCR reaction but vary the annealing temp in that reaction by 1oC: These reactions with different annealing temp are set up in separate tubes and require a PCR block with the ability to vary JUST the annealing temp in a reaction. I usually go by the lower of two Tm's.Laurence Stuart Dawkins-Hall Popular answerĪs somebody who is well versed and practiced in both gradient PCR and touch down PCR in essence, the answer is no: Both are troubleshooting versions of the standard 3 cycle PCR reaction Also, remember that the definition of Tm for a primer is "half of the primer is bound". By starting at a very high and stringent annealing temperature, you expect in the first few cycles to get a minimum amount of amplification of the exact product, then as the annealing temperature lowers in later cycles, there may be amplification of other products, but due to (A) the exponential nature of amplification, and (B) the fact that you've given the correct product a "head start", you hope that the correct product will out-amplify the off-target ones.įor sequencing, I'd think you'd want specificity over yield. I consider touchdown more to be about increasing yield in a messy amplification (like for a gel extraction). You can then pick a temperature that had the least amount of other junk. Those are more likely to be non-specific products. You can often see which bands disappear at higher temperatures compared to lower. I feel like gradient gives more information and more specificity. Anyone else, please feel free to correct me!) (I'm typing this out just as my understanding of the difference.
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