Fluorescence correlation spectroscopy of enzymatic DNA polymerization.

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.