title: Real-time detection of polymerase activity using supercritical angle fluorescence.
abstract: We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.
authors.affiliation: Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Krieg
authors.forename: Thomas
authors.initials: T
authors.lastname: Ruckstuhl
authors.forename: Stephan
authors.initials: S
authors.lastname: Laib
authors.forename: Stefan
authors.initials: S
authors.lastname: Seeger
mesh_terms: D015374:Biosensing Techniques; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D004259:DNA-Directed DNA Polymerase