DETECTION OF ASYMMETRIC PCR PRODUCTS IN HOMOGENEOUS SOLUTION BY FLUORESCENCE CORRELATION SPECTROSCOPY

The yield of the double-stranded DNA product (500 bp) of asymmetric PC R with a rhodamine-labeled primer (Rho-primer) was determined in a hom ogeneous solution using fluorescence correlation spectroscopy (FCS). F CS provides the average number of molecules in a focused volume and th e diffusion constant that relates the molecular weight. Since FCS meas ures the fluctuation of fluorescence intensity in a very small sample volume, the reaction mixture was directly placed on the FCS optical fi eld without any purification procedure after amplification. The result of changing the initial number of templates suggested that elongation of the Rho-primer could be detected by FCS in a PCR mixture containin g a single copy of the target gene in the initial condition. Possible scientific applications and perspectives of the proposed approach are discussed.