Real-time detection of nucleotide incorporation during complementary DNA strand synthesis |
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Authors: | Krieg Alexander Laib Stephan Ruckstuhl Thomas Seeger Stefan |
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Affiliation: | Physikalisch-Chemisches Institut, Universit?t Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. sseeger@pci.unizh.ch |
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Abstract: | Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction. |
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Keywords: | biosensors DNA labeling fluorescent probes polymerase sequencing |
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