Real-time detection of nucleotide incorporation during complementary DNA strand synthesis.
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.
Krieg
Alexander
A
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. sseeger@pci.unizh.ch
Laib
Stephan
S
Ruckstuhl
Thomas
T
Seeger
Stefan
S
D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism*; D013698:Templates, Genetic
D002232:Carbocyanines; D018076:DNA, Complementary; D003845:Deoxycytosine Nucleotides; D009841:Oligonucleotides; C085321:cyanine dye 5; D004256:DNA Polymerase I