For TD PCR, it's recommended that Ta be 10°C higher than the normal cycling Ta for the first 10 cycles or so, dropping 0.5 to 1.0°C/cycle. Should the extension temperature match this pattern to prevent miss-annealing? It seems that if the initial Ta was 75°C, then Text was set to 72°C (or whatever is optimal for the polymerase), the purpose of the high Ta would be lost.
Instead, should Text match Ta with an increased extension time until the optimal Text is reached?
the extension temperature should always be constant for your enzyme ( usually 72c) because that is the temperature where the enzyme makes fewest incorporation errors. TD logic is based on the thought that the primers are not ideal and will anneal to many sequences on the genome which will give a messy PCR. If you anneal at higher temperatures slowly coming down to the ideal annealing temperature then only the most specific priming will take place at the higher temperatures which will enrich the reaction with the most specific products early on in the cycling and give them a big advantage because they will amplify earlier than the similar sequences which anneal badly to the primers
Thanks Paul, and yes, the logic makes sense to me. However my question is for the case where the TD-PCR is to begin cycling with the annealing temperature above the ideal extension temperature (usually 72°C). Should the extension temperature remain constant during each cycle of this phase then, while the anneal temperature drops each cycle? It seems that if this is the case, the benefit of excluding nonspecifically annealed primers by annealing at a high temperature would be lost, since the primers may missanneal and be amplified during the lower temperature extension step.
Hi Matthew in essence no. Your extension temp should always remain constant as mentioned by Paul. In essence specificity is conferred by the primers and thus the annealing temp. It is only this phase of the PCR cycle that specifically alters during TD PCR. everything else should remain the same for a basic TD
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