Is it necessary to increase annealing T during PCR when there are adaptors in primers?

Question

[liuyifan2014]

Hi everyone,

I am thinking about it these days. I have a pair of primers. Both of them contain adaptor and gene-specific sequences(the true primer) while the reverse primer contains a index. Basically, those adaptors are added for binding to flow cell/glass lane for illumina sequencing. And obviously the Tm for these assembled primer is different from that for gene-specific sequences alone. My question is 'do we need increase annealing temperature during PCR?" As we all know, during the initial two rounds of PCR only gene-specific region binds to template DNA. After that the whole primer will bind to the assembled DNA. So I would like to set Tm to a low temperature in the first two cycle and set it to a higher temperature after that. Is it necessary?

Looking forward to hearing your thoughts! Yifan

Answer 1

[Anima Mundi]

Hello, maybe your question is not pertinent here. Yes, you might need to do that and in general it is not a bad practice. I would also start with something similar, 2-3 cycles with Ta adjusted to your primers without their tails, the rest with Ta adjuster for the whole primer length. In general, you also want Ta to accommodate the primer with a lower Tm so, if there is a strong difference in Tm between you full-length F primer and your full-length R primer, you might end-up not rising the Ta that much after the initial step.