Figure 5.
FRET in Flu-(dG)12–(dC)12-TAMRA during extension by Klenow exo−. (A) Time course of Flu emission. Polymerase extension assay was performed as described in Materials and Methods with 5 μM Flu-(dG)12–(dC)12-TAMRA and 0.8 μg/ml of Klenow exo−. The assay mixture containing Flu-(dG)12–(dC)12-TAMRA and nucleotides was transferred into a fluorimetric cuvette. Fluorescence emission at 520 nm was recorded in time as described in Materials and Methods; excitation was at 490 nm. A significant amount of energy transfer is detected as a large decrease in the contribution of the Flu donor and an increase in the contribution of the TAMRA acceptor. The extension reaction was started by addition of the enzyme and fluorescence was recorded in time. (B) Aliquots of 0.5 ml of sample was withdrawn from the incubation before (curve 1) and 5 (curve 2), 10 (curve 3), 20 (curve 4), 30 (curve 5), and 40 (curve 6) min after addition of the enzyme to the assay. The samples were passed through Sephadex G-25 DNA-Grade column (1 × 5 cm) in 20 mM Tris–Acetate buffer, pH 8.0, to separate high-molecular weight products of the synthesis from nucleotides; absorption spectra of the synthesized polymer eluted in the column's void volume were recorded. (C) The amount of G–C base pairs in double-labeled product of the synthesis were estimated from analysis of the spectra presented in (B) as described in Materials and Method, and plotted as a function of time of synthesis.