title: Exonucleolytic degradation of high-density labeled DNA studied by fluorescence correlation spectroscopy.
abstract: The exonucleolytic degradation of high-density labeled DNA by exonuclease III was monitored using two-color fluorescence correlation spectroscopy (FCS). One strand of the double stranded template DNA was labeled on either one or two base types and additionally at one end via a 5' Cy5 tagged primer. Exonucleolytic degradation was followed via the diffusion time, the brightness of the remaining DNA as well as the concentration of released labeled bases. We found a hydrolyzation rate of about 11 to 17 nucleotides per minute per enzyme (nt/min/enzyme) for high-density labeled DNA, which is by a factor of about 4 slower than for unlabeled DNA. The exonucleolytic degradation of a 488 base pair long double stranded DNA resulted in a short double stranded DNA segment of 112 ± 40 base pairs (bp) length with two single-stranded tails.
authors.affiliation: Institute of Physics, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. ehrlich@nano.ku.dk
authors.forename: Nicky
authors.initials: N
authors.lastname: Ehrlich
authors.forename: Katrin
authors.initials: K
authors.lastname: Anhalt
authors.forename: Hauke
authors.initials: H
authors.lastname: Paulsen
authors.forename: Susanne
authors.initials: S
authors.lastname: Brakmann
authors.forename: Christian G
authors.initials: CG
authors.lastname: Hübner
mesh_terms: D003116:Color; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III