Characterization of the 5
0
to 3
0
nuclease activity of Thermus aquaticus DNA
polymerase on fluorogenic double-stranded probes
Qiuying Huang, Qingge Li
*
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
article info
Article history:
Received 11 December 2008
Accepted 9 April 2009
Available online 17 April 2009
Keywords:
Taq DNA polymerase
5
0
-Nuclease
Real-time PCR
Double-stranded probes
Displacing probes
MALDI-TOF MS
abstract
Taq DNA polymerase contains a polymerase domain for synthesizing new DNA strands and a 5
0
-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
0
-nuclease activity is substrate dependent and its consequences remain largely unexplored, except
for its role in 5
0
-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
0
-nuclease activity of Taq DNA polymerase on fluorogenic double-stranded probes of varied struc-
tures. We observed that double-stranded probes with destabilized 5
0
-terminal could be hydrolyzed, and
the major cleavage was the removal of the 5
0
-terminal fluorophore-labeled nucleotide. These observa-
tions can serve as guidance for better design of double-stranded probes with reduced or no interfering
background for real-time PCR detection.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Thermus aquaticus (Taq) DNA polymerase is one of the most
important molecular biological tools widely used for PCR and
sequencing. Structural and functional analyses have revealed two
distinct domains in Taq polymerase, an N-terminal domain
(residues 1–289) that has single-stranded 5
0
-nucleotide cleavage
activity (5
0
-nuclease domain), and a C-terminal domain (residues
306–832) that has DNA polymerase activity [1]. The polymerase
domain and the 5
0
-nuclease domains are connected by a linker
region (residues 290–305). Exploration of its novel activities, as
well as fine tuning of these activities, has enabled the introduction
of new and improved applications for Taq DNA polymerase. For
example, the 5
0
to 3
0
nuclease activity of Taq DNA polymerase has
been employed in PCR to generate a specific detectable signal
concomitantly with amplification, resulting in the emergence of
real-time PCR [2], and the so-called 5
0
-nuclease probe or TaqMan
probe [3].
In real-time PCR, a variety of fluorophore-labeled oligonucle-
otide probes have been employed to generate sequence-specific
signals for quantitative evaluation. While TaqMan probes have to
be hydrolyzed during PCR by the exonucleolytic activity of Taq
DNA polymerase to generate a signal, other probes, such as
molecular beacons [4] and adjacent probes [5], need not be
hydrolyzed. Indeed, hydrolysis of these probes impairs the
performance of real-time PCR. For example, if adjacent probes are
hydrolyzed by 5
0
-nuclease activity, signal intensity is reduced due
to a successive reduction in the amount of intact probes, affecting
accuracy and sensitivity [6]. We have reported a new class of
nucleic acid probes, named ‘‘displacing probes’’ for homogeneous,
real-time PCR [7]. One distinct property of this kind of fluorogenic
double-stranded probes is their extremely high specificity, which
has been exploited in real-time PCR genotyping of a variety of
single nucleotide mutations [8–10] and has recently been further
improved by flexible modification approaches [11,12]. However,
we have occasionally found that in some displacing probe-based
real-time PCR assays the signal intensity increases linearly in the
absence of target. Such background signal can cause distortion of
the amplification curves, and occasionally results in failed
readout. The reason for such sporadic high-background fluores-
cence is unknown.
In the present work, we aimed to elucidate why only some
displacing probes induce nonspecific signals, while others do not.
Using both kinetic studies and MALDI-TOF MS analysis, we found
that nonspecific signals are caused by the 5
0
to 3
0
nuclease activity
of Taq DNA polymerase. In addition, by studying the effect of
*
Corresponding author. Tel./fax: þ86 592 2187363.
E-mail address: qgli@xmu.edu.cn (Q. Li).
Contents lists available at ScienceDirect
Molecular and Cellular Probes
journal homepage: www.elsevier.com/locate/ymcpr
0890-8508/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mcp.2009.04.002
Molecular and Cellular Probes 23 (2009) 188–194