Abstract
In a PCR-based 5' nuclease assay with a fluorogenic 16S rDNA probe, we found an increase in fluorescence that was not caused by the digestion of the DNA probe in the PCR. The fluorescence diminished to a base line level by ethanol precipitation of the 5' nuclease PCR product. Boiling the PCR product also decreased the fluorescence to the level of a control containing no template DNA. Hence, the increase in fluorescence in the assay did not seem to be caused by hybridization to the complementary structure of the template DNA, but seemed to be dependent on the intact probe remaining in the PCR reaction mixture. The increase in fluorescence appeared to be the result of a probe/template association other than the hybridization.
