A quantitative fluorescence-based steady-state assay of DNA polymerase.

Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye Pico Green. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo⁻, where we determine k_cat and K_m values for the DNA polymerase that are in excellent agreement with literature values. [ABSTRACT FROM AUTHOR]