Real-time PCR (qPCR) is the gold-standard for sensitive, specific detection and quantification of nucleic acid targets through fluorescence-based techniques.
TaqMan assays and SYBR Green dye are two popular approaches, each offering distinct advantages depending on a lab’s needs. By understanding these distinctions, researchers can select the approach that best aligns with their experimental goals, ensuring efficiency and precision.
This infographic explores key differences to help researchers determine the most suitable approach for their experimental workflows.
Download this infographic to:
- Explore differences in detection methods, assay components and signal generation
- Understand the impact of assay specificity on target and nontarget detection
- Compare workflow design and optimization for real-time PCR experiments
Signal
generation
Assay
components
Detection
method
Comparing SYBR Green I dye and
TaqMan Assays for real-time PCR analysis
Real-time PCR instruments detect fluorescent signals generated during target amplification. Thermo Fisher Scientific
offers products for two distinct detection chemistries that are widely used in research for fluorescence-based analysis of nucleic
acids by real-time PCR.
Dye-based detection: Invitrogen™ SYBR™ Green I dye
binds nonspecifically to double-stranded DNA, then
fluoresces upon excitation.
SYBR Green I dye produces a fluorescent signal
whether bound to the amplified target sequence,
nonspecific amplification products, or primer-dimers.
Target Nontarget Primer-dimer
SYBR Green dye–based assays include a forward
primer and a reverse primer designed to amplify a
specific target sequence. The dye is added to the
reaction separately, usually as a component of a
master mix.
Primer
Primer
Probe-based detection: Sequence-specific Applied
Biosystems™ TaqMan™ Assay probes are labeled with
a fluorescent reporter dye and a quencher.
During polymerization, the 5´ nuclease activity of Taq
DNA polymerase cleaves the bound probe. Once
separated from the quencher,
the reporter dye emits a
permanent fluorescent signal.
TaqMan Assays for gene expression analysis include
two PCR primers and a gene-specific TaqMan probe.
Reporter dye
5´ 3´
Quencher
Primer Probe
Primer
Reporter dye
5´ 3´
Quencher
Primer Probe
Primer
Reaction chemistry
SYBR Green I dye vs. TaqMan Assays
R Q
Primer
Target
When to use
Suitable for applications with low
specificity demands:
Mycoplasma infection in cell culture
Next-generation sequencing (NGS)
library quantification
Telomere length
Chromatin immunoprecipitation (ChIP)
Ideal for applications with high
specificity demands:
Gene expression analysis
miRNA analysis
Pathogen detection or quantification
Copy number variation, SNP genotyping
Clinical research
Assay design and
optimization
Multiplexing
Recommended
reagents
Ability to exclude
nontargets
Predesigned assays are not available for gene
expression analysis using SYBR Green I dye.
Developing an assay typically requires user design
and experimental optimization.
No
Applied Biosystems™ PowerTrack™ SYBR™
Green Master Mix
Thermo Fisher Scientific offers over 2.8 million verified,
ready-to-use TaqMan Gene Expression Assays for
research, with guaranteed* performance.
* Terms and conditions apply. To see full details of the guarantee, go to
thermofisher.com/taqmanguarantee.
Yes
Applied Biosystems™ TaqMan™ Fast Advanced
Master Mix or TaqMan™ Fast Virus 1-Step
Master Mix
TaqMan probes help
ensure nontargets are
excluded. Mismatched
probes are displaced
from homologs without
being cleaved, so no
signal is produced.
TaqMan probes cannot
bind to nonspecific PCR
products (e.g., primerdimers) and become
cleaved, so no signal is
produced.
Homolog
5´ 3´
Probe
Primer-dimer
5´ 3´
Probe
SYBR Green I dye vs. TaqMan Assays
Workflow design and optimization
For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All
trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of
Roche Molecular Systems, Inc., used under permission and license. TCN-8770498 1024
Learn more about real-time PCR products and applications at
thermofisher.com/qpcr
Ability to
detect target
Assay specificity
vs. TaqMan Assays
The specificity of an assay is its ability to detect targets and exclude nontargets
in samples. The use of 3 oligonucleotides in TaqMan Assays helps to ensure
higher specificity than the 2 oligonucleotides used in SYBR Green dye–based
assays.
Target detection relies on 2 primers, which must be
optimized for specificity.
Primer
Primer
Primer
Primer
Design 1 Design 2
Target detection relies on 2 primers and cleavage of
the bound sequence-specific probe, helping ensure
higher specificity.
Taq DNA
polymerase
Polymerase
domain
Nuclease
domain
TaqMan Primer probe Primer
Target Target
SYBR Green I dye
Signals from nontargets cannot be excluded. Post-run
specificity monitoring is recommended, but because
of inherent limitations it does not substitute for
specificity verification.
Melt curve analysis
Gel electrophoresis
Sequencing