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
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Secondly, the melting temperature isn't the absolute temperature beyond
which no DNA is bound; it's calculated as something of a midway point
between "No dissociation" and "All primers dissociated", so there's a
lot of wiggle-room. I also imagine that once a primer has bound, it has
a certain intrinsic stability that helps keep it there a while longer.
Of course, longer primers usually still work fine and have a higher Tm.
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics