FLUORESCENCE CORRELATION SPECTROSCOPY OF ENZYMATIC DNA POLYMERIZATION

We show that fluorescence correlation spectroscopy (FCS) can be used a s a reliable, simple, and fast tool for detecting products of the poly merase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DMA 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 rest riction enzymes. A FCS calibration curve is presented, where the trans lational diffusion times of different size DNA fragments are plotted v ersus the number of base pairs they contain. At zero and very low temp late concentrations a large ''background'' species emerges, which is a reflection of the experimental conditions chosen and the extremely hi gh sensitivity of FCS. The relative amount of nonspecific product form ation is less than 1%. The ease by which a FCS measurement can be perf ormed (a few minutes at most) also enables the technique to be used as an effective screening method.