Characterization of the 5' to 3' nuclease activity of Thermus aquaticus DNA polymerase on fluorogenic double-stranded probes

Mol Cell Probes. 2009 Jun-Aug;23(3-4):188-94. doi: 10.1016/j.mcp.2009.04.002. Epub 2009 Apr 17.


Taq DNA polymerase contains a polymerase domain for synthesizing new DNA strands and a 5'-nuclease domain for cleaving damaged DNA strands or RNA primers. Both of these domains play key roles in nucleic acid amplification and detection, especially in fluorogenic probe-based, real-time PCR. However, the 5'-nuclease activity is substrate dependent and its consequences remain largely unexplored, except for its role in 5'-nuclease-based TaqMan assays. Using both kinetic studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we comprehensively examined the 5'-nuclease activity of Taq DNA polymerase on fluorogenic double-stranded probes of varied structures. We observed that double-stranded probes with destabilized 5'-terminal could be hydrolyzed, and the major cleavage was the removal of the 5'-terminal fluorophore-labeled nucleotide. These observations can serve as guidance for better design of double-stranded probes with reduced or no interfering background for real-time PCR detection.

MeSH terms

  • DNA Probes / genetics
  • DNA Probes / metabolism*
  • Kinetics
  • Polymerase Chain Reaction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Taq Polymerase / metabolism*


  • DNA Probes
  • Taq Polymerase