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Cat. No.: PQMDO-100 (for 100T)

Cat. No.: PQMDO-500 (for 500T)

Description

 Probe qPCR Master Mix with UDG (One-Tube Premix) is a high-quality, contamination-prevention premix designed for probe-based real-time quantitative PCR (qPCR), including TaqMan probe-based detection. It is primarily intended for highly sensitive and specific singleplex or multiplex quantitative PCR detection of cDNA, genomic DNA, and other DNA templates.

This product supports the premixing of primers and probes to prepare a complete reaction mixture (ONE TUBE), which can be stored at low temperatures for extended periods. For subsequent use, the premixed reaction mixture can be added directly to the sample to be tested. The formulation contains optimized proportions of high-quality uracil-DNA glycosylase (UDG) and dUTP to effectively prevent false-positive results or artificially reduced Ct values caused by carryover contamination from previous PCR amplification products. Since TaqMan probes are commonly used in this detection method, it is also frequently referred to as UDG-mediated contamination-prevention TaqMan qPCR.

Components

• Probe qPCR Mix (5X, UDG)
• Ultrapure Water
• Low ROX (50X)
• High ROX (50X)

UDG-Mediated Carryover Contamination Prevention

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between uracil (dU) and deoxyribose in DNA strands containing uracil, releasing free uracil. UDG is widely used to prevent carryover contamination from previous PCR amplification products.

The contamination-prevention mechanism is as follows: an appropriate amount of dUTP is added to the PCR reaction to replace dTTP during DNA synthesis, resulting in PCR amplification products containing dU residues. During subsequent PCR reactions, UDG selectively cleaves uracil-containing DNA carried over from previous PCR amplifications, preventing these contaminants from adversely affecting the current PCR reaction.

Applications

Probe-based qPCR is particularly suitable for the quantitative or qualitative detection of low-abundance targets and targets requiring high specificity.

Typical applications include:

• Highly sensitive detection of low-abundance mRNA, lncRNA, and small RNA following reverse transcription.

• Detection of microbial RNA following reverse transcription.

• Discrimination of homologous genes with high sequence similarity that may be difficult to distinguish using conventional dye-based qPCR.

• Detection of single-nucleotide polymorphisms (SNPs).

• Singleplex and multiplex real-time PCR assays requiring high specificity and sensitivity.

Detection Principle of Probe-Based qPCR

Unlike dye-based qPCR methods that use fluorescent dyes such as SYBR Green to detect amplified DNA, probe-based qPCR uses sequence-specific DNA probes labeled with a fluorescent reporter and a quencher to detect the target sequence. The probe-binding site is typically located between the binding sites of the two PCR primers.

Under normal conditions, fluorescence from the reporter is quenched because of its proximity to the quencher through fluorescence resonance energy transfer (FRET). During amplification of the target sequence, both primers and the probe anneal to the target DNA. As the primers are extended, the 5′→3′ exonuclease activity of Taq DNA polymerase progressively degrades the probe bound to the target sequence, starting from its 5′ end.

When the fluorescent reporter and quencher are separated by probe cleavage, the quenching effect is eliminated, allowing the reporter to emit fluorescence upon excitation by the appropriate light source. With each PCR cycle, more fluorescent reporters are released. The fluorescence signal increases in proportion to the accumulation of amplified target DNA, enabling quantitative detection.

Probes are typically linear DNA oligonucleotides specific to the target sequence, labeled with a fluorescent reporter such as FAM or HEX at the 5′ end and a quencher such as BHQ1 or TAMRA at the 3′ end. MGB-containing probes may also be used, depending on the probe design.

Features

High Specificity and Sensitivity: Detection specificity depends not only on the PCR primers but also on the sequence-specific hybridization of the probe to the target. Fluorescence is generated when the probe binds to the target sequence and is cleaved during amplification. This additional sequence-recognition step generally provides greater specificity than dye-based methods using fluorescent dyes such as SYBR Green. Probe-based detection is particularly useful when distinguishing closely related target sequences or detecting low-abundance targets.

Multiplex qPCR Detection: Multiple targets can be detected in a single reaction well by using target-specific probes labeled with different fluorescent reporters. With appropriate optimization of primer and probe concentrations and compatibility with the instrument's fluorescence detection channels, this product can be used to simultaneously detect up to 4–5 genes. The actual number of targets that can be detected simultaneously depends on the assay design, fluorescent reporter combinations, instrument configuration, and reaction optimization.

Antibody-Mediated Hot-Start Taq DNA Polymerase: The Taq DNA polymerase used in this product is a high-quality antibody-mediated hot-start enzyme designed to provide convenient and efficient hot-start PCR. The Taq DNA polymerase is bound to a monoclonal anti-Taq antibody, which inhibits its DNA polymerase activity before the initial denaturation step. This helps prevent nonspecific amplification caused by nonspecific primer-template annealing and primer-dimer formation at low temperatures. During the initial denaturation step of PCR, the antibody is inactivated by heating, releasing the activity of Taq DNA polymerase. DNA amplification therefore begins after the initial denaturation step, improving PCR specificity, sensitivity, and quantitative accuracy. The upgraded Taq DNA polymerase also offers greater compatibility with different template types, template GC contents, and primer melting temperatures (Tm).

One-Tube Premix and Extended Storage Stability: The master mix contains the essential common reaction components, including Taq DNA polymerase, UDG enzyme, PCR buffer, dNTPs, dUTP, stabilizers, and magnesium ions. This simplifies reaction preparation and improves workflow convenience.Users only need to provide primers, probes, template DNA, and ultrapure water when preparing the reaction mixture. The specially optimized PCR buffer helps maintain Taq DNA polymerase stability during storage. The premixed reaction mixture can be stored under the following conditions:

• 37°C: Up to 1 week

• 4°C: Up to 2 months

• −20°C: More than 6 months

After storage, the premixed reaction mixture can be used by adding the sample to be tested, simplifying routine qPCR setup. The product is also compatible with fast PCR protocols, helping reduce overall detection time when appropriate cycling conditions are used. Actual storage performance may depend on the primer and probe sequences, formulation compatibility, storage conditions, and handling procedures. Storage stability should be validated for the specific premixed assay.

ROX Reference Dye Compatibility

This product is available in Low ROX and High ROX formulations to accommodate real-time PCR instruments that require different concentrations of ROX passive reference dye, as well as instruments that do not require ROX.

ROX is used to normalize fluorescence fluctuations unrelated to PCR amplification, helping minimize well-to-well variation. Such variation may arise from factors including pipetting errors and sample evaporation.

Different real-time PCR instruments have different ROX requirements. When preparing the reaction mixture, select the appropriate formulation according to the instrument being used: High ROX, Low ROX, or no ROX.

For optimal results, refer to the instrument manufacturer's recommendations regarding passive reference dye requirements.