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Cat. No.: PQPUT-100 (for 100T)

Cat. No.: PQPUT-500 (for 500T)

Cat. No.: PQPUT-2500 (for 2500T)

Description

Probe qPCR Mix with UDG and Tracking Dyes is a new high-quality carryover prevention premix used for real-time fluorescence quantitative PCR, specifically probe-based qPCR (Quantitative PCR) or Real-time PCR, utilizing TaqMan and other probes. It contains a dual-dye tracking system that uses the color change effect produced by mixing two tracking dyes to monitor the pipetting process. This helps users distinguish between blank wells and wells with qPCR Mix, and confirm whether small volumes of DNA samples have been added to the qPCR Mix. It can be quickly and conveniently used for highly sensitive quantitative detection of specific cDNA or genomic DNA. This product contains an optimized ratio of high-quality UDG enzyme and dUTP, effectively eliminating false positives or low CT values caused by product contamination during PCR amplification.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between uracil (dU) bases and deoxyribose in DNA strands containing uracil, releasing free uracil. It is mainly used to eliminate product contamination issues during PCR amplification. The principle of preventing contamination is to add an appropriate amount of dUTP to the PCR reaction, replacing dTTP with dUTP in the DNA, forming PCR amplification products containing dU bases. In subsequent PCR reactions, UDG enzyme selectively cleaves single-stranded or double-stranded DNA containing dU from previous PCR amplifications that may have been contaminated, thus avoiding the negative impact of previous PCR amplification products on the current PCR amplification.

When preparing the qPCR reaction system, the volume of DNA samples added is usually only about 1-2 microliters, making it difficult to visually confirm whether the sample has been added. The Probe qPCR Mix (2X, UDG, Blue) in this product contains an inert blue dye for tracking, and it also provides a Template Dilution Buffer (40X, Yellow) with an inert yellow dye. When preparing the qPCR reaction system, the solution color will change significantly as the two components are mixed. When the yellow solution is added to the blue solution, it turns green, allowing users to accurately determine whether the DNA template and qPCR mix have been added based on the color change. This tracking function improves the accuracy of setting up the qPCR system, avoiding missed or incorrect additions of the template.

This product is particularly suitable for quantitative or qualitative detection of low-abundance or highly specific target genes. For example, it can be used for highly sensitive detection of low-abundance mRNA, lncRNA, small RNA, and microbial RNA after reverse transcription, as well as for SNP detection of homologous genes that are difficult to distinguish using conventional dye methods.

The product has high specificity and sensitivity. Specificity relies not only on PCR primers but also on the specificity of the probe. The probe binds and degrades specifically with the target gene to generate a fluorescent signal. The detection sensitivity and specificity are usually significantly higher than methods using fluorescent dyes like SYBR Green.

This product can be used for multiplex detection. In a single reaction well, different genes correspond to different probes, and different probes correspond to different fluorescent labels, enabling multiplex fluorescence quantitative PCR detection. Tests have shown that with appropriate optimization of primers and probes, this product can be used to detect 2-3 genes simultaneously.

The product uses DNA probes labeled with a fluorescent group and a quencher group (Quencher) targeting the target sequence to be detected by PCR (the designed probe binding site is usually located between the binding sites of the two primers). Normally, the quencher group on the probe quenches the fluorescent group due to spatial fluorescence resonance energy transfer (FRET). During PCR amplification of the target sequence, both primers and probes anneal to the target gene. As the primer extends, the 5’→3’ exonuclease activity of Taq enzyme gradually degrades the probe bound to the target sequence from the 5’ end. Once the fluorescent group and quencher group on the probe are cleaved by Taq enzyme, the quencher group’s effect disappears, and the fluorescent group can be excited by light to produce fluorescence.

It has been repeatedly verified that the spectra of the blue and yellow inert tracking dyes added in this product do not overlap with the fluorescent group and quencher group, ensuring that they do not affect fluorescence quantitative detection or the enzyme activity of the amplification system. With each PCR cycle, more fluorescent groups are released, and the fluorescence intensity is proportional to the number of newly synthesized target fragments, enabling quantitative detection. The probe is usually a linear DNA specific to the target sequence, labeled with a fluorescent group such as FAM or HEX at the 5’ end, and a fluorescent quencher group such as BHQ1, TAMRA, or MGB at the 3’ end.

The Probe qPCR Mix (2X, UDG, Blue) in this product contains HS Taq DNA Polymerase, blue tracking dye, PCR buffer, dNTPs, stabilizers, and magnesium ions, making the operation simpler and more convenient. Users only need to prepare probes, primers, sample DNA, and deionized water.

The HS Taq DNA Polymerase used in this product is a high-quality hot-start enzyme combined with an antibody, enabling convenient and efficient hot-start. The Taq enzyme in HS Taq DNA Polymerase is bound to a monoclonal antibody against Taq, inhibiting the DNA polymerase activity of Taq. This effectively prevents non-specific amplification caused by non-specific annealing of primers and template DNA or primer-dimers at low temperatures. During the pre-denaturation step of the PCR reaction, the antibody is heat-inactivated, ensuring that the Taq enzyme activity is only released after pre-denaturation. This prevents DNA polymerization reactions before pre-denaturation, greatly improving the specificity, sensitivity, and accuracy of quantitative detection in PCR reactions.

This product offers Low ROX and High ROX, making it widely compatible with fluorescence quantitative PCR instruments that do not require ROX or need Low ROX or High ROX as a reference dye. ROX is used to correct fluorescence fluctuations unrelated to PCR, minimizing well-to-well differences. These differences can be caused by various factors such as pipetting errors and sample evaporation. Different fluorescence quantitative PCR instruments have different requirements for ROX. Please choose high concentration ROX (High ROX), low concentration ROX (Low ROX), or no ROX according to the actual instrument used when preparing the reaction system. Typically, Probe One-Step qRT-PCR Kit with high concentration ROX (with tracking dye) can also be used for fluorescence quantitative PCR instruments that do not require ROX or need low concentration ROX.

Components

• Probe qPCR Mix (2X, UDG, Blue)
• Template Dilution Buffer (40X, Yellow)
• Low ROX (50X)
• High ROX (50X)

Features

• This product has good stability and can be stored at 37ºC for 3 days without affecting its amplification performance.
• Compared to similar products, this product has more significant color differences and more sensitive amplification performance.