Determination of the reopening temperature of a DNA hairpin structure in vitro.

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5- and 3'-termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3'-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3'-end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.