Assay of DNA denaturation by polymerase chain reaction-driven fluorescent label incorporation and fluorescence resonance energy transfer.

We have developed a simple and sensitive assay method to monitor DNA denaturation. This method is based on (i) incorporation of fluorescent labels onto complementary DNA strands during target-specific amplification by polymerase chain reaction and (ii) detection of DNA denaturation by fluorescence resonance energy transfer. Fluorescein-11-dUTP and rhodamine-4-dUTP were used to label complementary DNA strands as the energy transfer donors and acceptors, respectively. The complementary DNA strands thus labeled were then annealed by incubation at a constant temperature, and the energy transfer efficiency was monitored during DNA denaturation experiments. Both heat denaturation and alkali denaturation could be monitored by this assay. Some factors affecting the DNA denaturation were also investigated.