At Takara Bio, great enzymes are part of our DNA. With over 90 years of experience in the pursuit of excellence in biochemistry and molecular biology technologies, we have developed an extensive portfolio of tools that have been widely published in peer-reviewed studies. Explore below to see how we can solve your challenges with probe-based quantitative PCR.
Advantages of probe-based qPCR
High specificity, sensitivity, and reproducibility
Compatible with multiplexing
Ideal for genotyping and copy number variation (CNV) analyses
Each sequence requires design and optimization for different probes
Quantitative PCR (qPCR) is a powerful and common technique for accurate analysis of gene expression. Our probe-based qPCR kits are optimized for accurate target quantification and detection over a broad dynamic range, enabling highly reproducible qPCR. These premixes are compatible with all fluorescent oligonucleotide probes or 5' nuclease assays. When deciding whether to do probe-based qPCR or intercalating dye-based qPCR, it is important to consider the advantages and limitations of each method.
Often, probe-based qPCR is used for clinical and diagnostic applications, as it is well-suited for any application that requires maximum specificity and consistent performance. Probe-based qPCR can attain this level of performance because the probes are highly specific for the target sequences and the formation of primer dimers is very rare. Since each probe is uniquely labeled, multiple targets can be amplified in the same tube via multiplexing. This increases throughput, minimizes sample handling, and potentially decreases reagent and consumable usage. Finally, because of the sensitivity and capability of multiplexing, probe-based qPCR works well for genotyping and CNV analyses because the probes can accurately discriminate between different single nucleotide polymorphisms (SNPs) or CNVs.
However, probe-based qPCR has some limitations. While the probes are highly specific, each individual probe can require multiple iterations of design and optimization steps to ensure maximum specificity and performance. Additionally, as the probe is not just an oligonucleotide but contains a fluorophore and a quencher, the probes themselves are often more expensive than simple oligonucleotide primers. While predesigned probes are available, these often have even higher costs.
We offer multiple formats of probe-based qPCR mixes to meet your experimental needs and give you the flexibility to perform a wide variety of applications.
Principle of probe-based qPCR
Oligonucleotides modified with a 5’ fluorophore (e.g., FAM) and a 3’ quencher (e.g., TAMRA) are added to the reaction. Under annealing conditions, the probe hybridizes in a sequence-specific manner to the template DNA. Fluorescence of the fluorophore is suppressed by the quencher. During the extension reaction, the 5’→ 3’ exonuclease activity of Taq DNA polymerase degrades the hybridized probe, releasing quencher suppression and allowing fluorescence (Figure 1). To maximize sensitivity, our kits use TaKaRa Ex Taq DNA Polymerase Hot-Start Version, a hot-start PCR enzyme that minimizes nonspecific amplification that may arise from mispriming or primer-dimer formation during reaction mixture preparation and pre-cycling steps.
Probe-based qPCR using Ex Taq master mix
Premix Ex Taq (Probe qPCR) is a 2X master mix for probe-based qPCR or 5' nuclease assays. This 2X master mix includes TaKaRa Ex Taq DNA Polymerase Hot-Start Version—a hot-start PCR enzyme combined with an anti-Taq antibody—in a qPCR-optimized buffer.TaKaRa Ex Taq DNA Polymerase Hot-Start Version inhibits nonspecific amplification while enabling high-efficiency amplification and detection sensitivity during real-time PCR analyses. Additionally, a heat-resistant Tli RNase H is included in the real-time PCR premix in order to minimize PCR inhibition due to the presence of residual mRNA in the input cDNA. This master mix is ideal for high-speed PCR, since it allows accurate target quantification and detection over a broad dynamic range, enabling highly reproducible and reliable qPCR analyses.
We provide our probe-based qPCR kits in three formats to suit your experimental needs:
Flexibility: Premix Ex Taq (Probe qPCR), Cat. # RR390A/B comes with two different tubes of ROX dyes for maximum instrument support
Throughput: Premix Ex Taq (Probe qPCR), Cat. # RR390L/W comes in volumes up to 25 ml with one ROX dye tube and is ideal for bulk applications
Convenience: Premix Ex Taq (Probe qPCR), Cat. # RR390LR/WR comes premixed with ROX dye and is available in small and large volume formats (up to 25 ml)
Here are a few examples of research that's been driven by our probe-based qPCR kits:
Jiang, Y. et al. Aberrant expression of RSK4 in breast cancer and its role in the regulation of tumorigenicity. Int. J. Mol. Med.40, 883–890 (2017).
Cat. # RR390A was used to accurately and specifically detect altered expression of RSK4 in multiple cell and tissue samples.
Jordan, J. A. & Durso, M. B. Real-time polymerase chain reaction for detecting bacterial DNA directly from blood of neonates being evaluated for sepsis. J. Mol. Diagn.7, 575–81 (2005).
Cat. # RR390A was used to develop a rapid assay capable of detecting bacterial DNA directly from blood samples.
Kim, H.-R. et al. Multiplex real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3. J. Virol. Methods250, 11–16 (2017).
Cat. # RR390A was used to develop a multiplex qPCR assay for the rapid and differential detection of porcine circovirus 2 and 3. This sensitive and reproducible assay had a limit of detection below 50 copies and achieved coefficients of intra-assay and inter-assay variation of less than 4%.
Teng, Q. et al. Development of a TaqMan MGB RT-PCR for the rapid detection of H3 subtype avian influenza virus circulating in China. J. Virol. Methods217, 64–69 (2015).
Cat. # RR390A was used with minor groove binder probes to develop a sensitive and rapid assay for H3 avian influenza virus. This assay was 1,000X more sensitive than conventional qPCR, with a detection limit of 10 copies per reaction.
High-throughput real-time PCR
Where throughput meets flexibility
Real-time PCR (qPCR) is a powerful technique for genotyping and gene expression analysis. Currently, qPCR experiments are becoming increasingly complex—involving an expansive and growing list of targets from a larger number of samples, all with more technical replicates. The SmartChip Real-Time PCR System is a complete high-throughput solution that enables an unrivaled amount of flexible assay and sample formats, allowing researchers to seamlessly switch between dispensing assay reagents and samples into blank chips, or dispensing samples into custom, preprinted chips without the need for revalidation.