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Cat. No.: PQU-N-1 (for 1ml, No ROX)

Cat. No.: PQU-N-5 (for 5ml, No ROX)

Cat. No.: PQU-N-25 (for 25ml, No ROX)

Cat. No.: PQU-L-1 (for 1ml, Low ROX)

Cat. No.: PQU-L-5 (for 5ml, Low ROX)

Cat. No.: PQU-L-25 (for 25ml, Low ROX)

Cat. No.: PQU-H-1 (for 1ml, High ROX)

Cat. No.: PQU-H-5 (for 5ml, High ROX)

Cat. No.: PQU-H-25 (for 25ml, High ROX)

Description

Probe qPCR Mix (2X, UDG) is a new high-quality carryover prevention premix for real-time fluorescent quantitative PCR based on probes such as TaqMan. It is designed for ultra-sensitive specific quantitative detection of cDNA and genomic DNA, among others. This product contains an optimized ratio of high-quality UDG enzyme and dUTP, effectively eliminating false positives or abnormally low CT values caused by contamination from PCR products. As the probe commonly used is the TaqMan probe, this method is also frequently referred to as the carryover prevention TaqMan probe method.

UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), catalyzes the hydrolysis of the N-glycosidic bond between the uracil (dU) base and deoxyribose in the DNA chain containing uracil, thus releasing free uracil. It is mainly used to eliminate contamination issues caused by PCR amplification. The mechanism for preventing contamination is: adding an appropriate amount of dUTP to the PCR reaction, incorporating dUTP instead of dTTP into the DNA to produce PCR amplification products containing the dU base. In subsequent PCR reactions, the UDG enzyme selectively cuts the single or double-stranded DNA that may have been contaminated from previous PCR amplification containing dU, thereby avoiding potential contamination effects on the current PCR amplification.

This product is particularly suitable for the quantitative or qualitative detection of low abundance or highly specific target genes. Examples include high sensitivity detection of low abundance mRNAs, lncRNAs, small RNAs, microbial RNA post-transcription, and SNP detection of homologous genes or genes that are hard to differentiate using conventional dye methods due to high similarity.

Principle

Probe-based qPCR does not use fluorescent dyes like SYBR Green to stain PCR products. Instead, it employs a DNA probe labeled with a fluorescent group and a quenching group (quencher) targeting the sequence intended for PCR detection (the designed probe binding site typically lies between the two primer binding sites). Normally, the quencher on the probe quenches the fluorescent group due to spatial fluorescence resonance energy transfer (FRET). During PCR reaction amplification of the target sequence, both the primer and the probe anneal to the target gene. As the primer extends, the 5'→3' exonuclease activity of the Taq enzyme results in the probe bound to the target sequence being gradually degraded from the 5' end. Once the Taq enzyme cleaves the fluorescent group and the quencher of the probe, the quenching effect disappears, and the fluorescent group can be excited by the excitation light to produce fluorescence. 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. Probes are typically a linear DNA sequence specific to the target sequence, labeled with a fluorescent group like FAM or HEX at the 5' end and a quenching group like BHQ1, TAMRA, or MGB at the 3' end.

Features

• High specificity and sensitivity: Specificity depends not only on the PCR primers but also on the specificity of the probe. Only when the probe specifically binds and degrades with the target gene will a fluorescent signal be generated. The detection sensitivity and specificity are generally much higher than methods using fluorescent dyes like SYBR Green.
• For multiplex detection: in a single reaction well, different genes correspond to different probes, and different probes correspond to different fluorescent labels, enabling multiplex fluorescent quantitative PCR detection. Testing has shown that after appropriate optimization of primers and probes, this product can be used to detect 2-3 genes simultaneously.
• Hotstart property: The Hotstart Taq DNA Polymerase used in this product is a high-quality heat-start enzyme bound to an antibody. It allows for convenient and efficient heat activation. In the Hotstart Taq DNA Polymerase, the Taq enzyme binds with a monoclonal antibody against the Taq enzyme, inhibiting its DNA polymerase activity, which effectively prevents non-specific annealing of primers and template DNA or primer dimerization at low temperatures. The antibody is heat-inactivated during the pre-denaturation step of the PCR reaction, ensuring that Taq enzyme activity is only released after pre-denaturation, avoiding DNA polymerization reactions before pre-denaturation, greatly enhancing the specificity, sensitivity, and quantitative detection accuracy of the PCR reaction.
• Easy to use: This product includes all common components like Hotstart Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, stabilizers, and magnesium ions, making the process simpler and more convenient for users. Users only need to prepare primers, probes, sample DNA, and deionized water.

ROX Dye

The role of ROX dye is to correct fluorescence fluctuations unrelated to PCR, thereby minimizing inter-well variability. This variation might be caused by factors such as pipetting errors and sample evaporation. Different quantitative PCR instruments have varying requirements for ROX. Depending on the specific instrument in use, one should choose a Probe qPCR Mix (2X, UDG) containing either a high concentration of ROX (High ROX), a low concentration of ROX (Low ROX), or no ROX. Typically, the Probe qPCR Mix containing a high concentration of ROX can also be used for fluorescence quantitative PCR instruments that don't require ROX or require a low concentration of ROX.

Storage

Precautions

• The choice of fluorescence label for the probe should be determined based on the fluorescence compatibility of the qPCR instrument being used. For fluorescent quantitative PCR machines that require ROX as a correction dye, avoid using probes labeled with ROX.
• When used for multiplex detection, it is necessary to properly optimize the primers and probes, and use appropriately differently labeled fluorescence probes. After determining the results, proceed with multiplex detection, and it is generally recommended not to exceed triplex detection. If used for triplex or quadruplex detection, Multiplex Probe qPCR Mix (2X, UDG) is recommended.
• Before use, ensure that the entire tube of reagent is completely melted. Invert gently to mix before using. Try to avoid producing bubbles during the mixing process.
• Pay attention to the primer annealing temperature. When the annealing temperature is <60ºC, it is recommended to use a three-step PCR amplification.
• For amplification fragments exceeding 350bp or with a high GC content, it is suggested to extend the time to 60 seconds or use the three-step method to improve the amplification efficiency.
• It has been tested that the product can withstand up to 10 freeze-thaw cycles without significant impact on its efficacy. However, it is still advised to avoid repeated freeze-thawing as this could potentially degrade the product's performance.
• qPCR detection is an ultra-sensitive method. Although this product has excellent contamination prevention, the PCR reaction setup area should still avoid any potential contamination by amplicons. PCR products should be sealed and discarded to prevent high-concentration PCR products from contaminating the experimental environment.
• This product is intended for scientific research by professionals only. It should not be used for clinical diagnosis or treatment, and is not to be used in food or drugs, nor should it be stored in regular residences.
• For your safety and health, please wear a lab coat and disposable gloves while handling.

Related:

• Probe One-Step qRT-PCR Kit (20X, UDG)
• Multiplex Probe qPCR Mix (2X, UDG)
• Probe qPCR Mix (2X, UDG)
• SYBR Green One-Step qRT-PCR Kit (20X, UDG)
• SYBR Green qPCR Mix (2X, UDG)

Only for research and not intended for treatment of humans or animals

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