Quantitative analysis by the polymerase chain reaction using fluorescence correlation spectroscopy

Abstract

DNA amplification by the polymerase chain reaction (PCR) was monitored at the level of single molecules. The technique used consisted of a direct fluorescent labeling method using the PCR and measurement of fluorescence fluctuation by fluorescence correlation spectroscopy (FCS). An increasing number of target DNA molecules during amplification resulted 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. Increasing the apparent labeling density according to concentration of fluorescein-11-dUTP was evaluated from the fluorescence intensity per DNA molecule. The number of amplified DNA molecules could be detected quantitatively after 10 PCR cycles even when the initial template number was 3750 copies; however, a linear relationship between the initial template number and amplified DNA number was shown at 20 cycles in PCR.