Exonuclease degradation of DNA studied by fluorescence correlation spectroscopy

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and/or single-stranded DNA/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3' to 5' exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times. We give proof of: (i) complete enzymatic degradation, (ii) retardation of complete enzymatic degradation by internally labelled Rhodamine-4-nucleotides and Cy5-nucleotides, respectively. Data evaluation by global analysis indicated first-order reaction kinetics with full-length DNA and free fluorescent nucleotides in the time window of measurements used.