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