RE: primer Tm reasoning?

Jonathan;

You are not wrong..if you can design a primer that doesn’t produce a bunch of cross reactivities that has a high Tm..then so be it.

Signal Diagnostics has a patented technology termed Dynamic Flux Amplification, where we use annealing at temperatures much elevated from the conventional 50-65C range.  It works beautifully..

Brian

Sorry to start yet another new thread for such a small question, but I don't want to pollute other threads with off topic posts... I've been trying to understand why primer melting temperatures have to be in the 55-65C range when PCR cycles call for 94C for DNA melting... I've tried to find an explanation for this but haven't been able to... Wouldn't that result in the primers splitting off from the template DNA before you reach the extension cycle of 75C (or around there)? I'm sure I'm wrong because obviously these PCR programs work, but I don't know WHY I'm wrong, and would like to understand. Thanks again for explaining something that is probably so common knowledge that nobody feels the need to address it in writing :)

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