Exonucleolytic degradation of high-density labeled DNA studied by fluorescence correlation spectroscopyElectronic supplementary information (ESI) available. See DOI: 10.1039/c2an15879e

• Main Authors: Ehrlich, Nicky, Anhalt, Katrin, Paulsen, Hauke, Brakmann, Susanne, Hbner, Christian G
• Format: Article
• Language: English
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c2an15879e

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. The exonuclease III degradation of different labeled DNA-templates was characterized by two-color fluorescence correlation spectroscopy in respect to single-molecule DNA-sequencing.