Detection of asymmetric PCR products in homogeneous solution by fluorescence correlation spectroscopy

Abstract

The yield of the double-stranded DNA product (500 bp) of asymmetric PCR with a rhodamine-labeled primer (Rho-primer) was determined in a homogeneous solution using fluorescence correlation spectroscopy (FCS). FCS provides the average number of molecules in a focused volume and the diffusion constant that relates the molecular weight. Since FCS measures the fluctuation of fluorescence intensity in a very small sample volume, the reaction mixture was directly placed on the FCS optical field 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 containing a single copy of the target gene in the initial condition. Possible scientific applications and perspectives of the proposed approach are discussed.