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

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

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Department of Biomedical Sciences and the Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.


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.

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