QUANTITATIVE-ANALYSIS BY THE POLYMERASE-CHAIN-REACTION USING FLUORESCENCE CORRELATION SPECTROSCOPY

DNA amplification by the polymerase chain reaction (PCR) was monitored at the level of single molecules. The technique used consisted of a d irect fluorescent labeling method using the PCR and measurement of flu orescence fluctuation by fluorescence correlation spectroscopy (FCS). An increasing number of target DNA molecules during amplification resu lted in a decrease of the number fluctuations, and also an increase of the average diffusion time. Fluorescein-11-dUTP was incorporated into the DNA strand with a length of 4000 bp using Taq DNA polymerase. Inc reasing the apparent labeling density according to concentration of fl uorescein-11-dUTP was evaluated from the fluorescence intensity per DN A molecule. The number of amplified DNA molecules could be detected qu antitatively after 10 PCR cycles even when the initial template number was 3750 copies; however, a linear relationship between the initial t emplate number and amplified DNA number was shown at 20 cycles in PCR. (C) 1998 Elsevier Science B.V.