Concentration of dye-labeled nucleotides incorporated into DNA determined by surface plasmon resonance-surface plasmon fluorescence spectroscopy.

A method for the accurate determination of the fraction of surface-attached DNA duplexes exhibiting a single-fluorophore-labeled nucleobase is introduced. The fluorescence signals obtained from surface plasmon field-enhanced fluorescence spectroscopy along with the optical properties of the sensor architecture determined by surface plasmon resonance were employed for the calculation. A Cy5-labeled nucleotide was incorporated into DNA at a well-defined position via template-directed DNA synthesis performed by a DNA polymerase. The sample-to-sample variations associated with the optical properties of the employed metal films caused a small variation in the strength of the evanescent field. This variation was accounted for by evanescent field integration over the DNA layers. The exponential-type relationship between the fraction of DNA with Cy5-dCTP incorporation at the surface and the mole fraction of the Cy5-dCTP in solution indicates the preferential incorporation of nonlabeled nucleotides by the DNA polymerase.