New insights into the annealing behaviour of DNA

In mutagenesis experiments with megaprimers of length 450-770 bp optimal annealing of the megaprimer to the template-DNA was shown to occur at unexpectedly low temperatures under conditions suboptimal for amplification of the elongated megaprimer-product by the short flanking primers, although very high annealing temperatures would have been expected to give best results. This finding contradicts the rule that annealing temperatures should be just below the melting temperature of a given primer, and leads to the conclusion that the curves of melting and annealing temperatures diverge rather than running in parallel.