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Real‐time Detection of Nucleotide Incorporation During Complementary DNA Strand Synthesis
Author(s) -
Krieg Alexander,
Laib Stephan,
Ruckstuhl Thomas,
Seeger Stefan
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200200549
Subject(s) - klenow fragment , nucleotide , dna , chemistry , dna synthesis , fluorescence , polymerization , nucleobase , biophysics , stereochemistry , combinatorial chemistry , biochemistry , dna polymerase , biology , gene , exonuclease , organic chemistry , physics , quantum mechanics , polymer
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.