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