Real-time detection of nucleotide incorporation during complementary DNA strand synthesis

Chembiochem. 2003 Jul 7;4(7):589-92. doi: 10.1002/cbic.200200549.


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.

MeSH terms

  • Base Sequence
  • Carbocyanines / chemistry
  • DNA Polymerase I / metabolism
  • DNA, Complementary / biosynthesis*
  • DNA, Complementary / chemical synthesis
  • DNA, Complementary / chemistry*
  • Deoxycytosine Nucleotides / chemistry
  • Deoxycytosine Nucleotides / metabolism
  • Fluorescence
  • Molecular Sequence Data
  • Nucleic Acid Hybridization / methods*
  • Oligonucleotides / chemistry*
  • Oligonucleotides / metabolism*
  • Templates, Genetic


  • Carbocyanines
  • DNA, Complementary
  • Deoxycytosine Nucleotides
  • Oligonucleotides
  • cyanine dye 5
  • DNA Polymerase I