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/oligonucle otides using fluorescent reporter dyes. 217-bp DNA fragments were prod uced by polymerase chain reaction and cleaved by the 3' to 5' exonucle ase activity of T7-DNA polymerase. The analysis of the products was pe rformed by Fluorescence Correlation Spectroscopy measuring autocorrela tion amplitudes and diffusion times. We give proof of: (i) complete en zymatic degradation, (ii) retardation of complete enzymatic degradatio n 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 nucleotid es in the time window of measurements used.