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Cat. No.: MPQU-100 (for 100T)
Cat. No.: MPQU-500 (for 500T)
Cat. No.: MPQU-2500 (for 2500T)
Description
Multiplex Probe qPCR Mix (2X, UDG) is a new high-quality anti-contamination (Carryover prevention) premix for multiplex real-time fluorescent quantitative PCR based on probes such as TaqMan. It is mainly used for ultra-sensitive specific quantitative detection of cDNA and genomic DNA. This product contains an optimized proportion of high-quality UDG enzyme and dUTP, which effectively eliminates the false positives or low CT values caused by product contamination during PCR amplification. Since TaqMan probes are generally used, this method is also commonly referred to as the anti-contamination multiplex TaqMan probe method.
UDG (Uracil-DNA Glycosylase), also known as UNG (Uracil-N-glycosylase), can catalyze the hydrolysis of the N-glycosidic bond between the uracil (dU) base in the DNA strand containing uracil and deoxyribose, thereby releasing free uracil. It is mainly used to eliminate product contamination problems caused by PCR amplification. Its contamination prevention principle is: by adding an appropriate amount of dUTP in the PCR reaction, replacing dTTP with dUTP in DNA, forming PCR amplification products containing dU bases. In subsequent PCR reactions, the UDG enzyme selectively cuts the single or double-stranded DNA containing dU that may have been contaminated from previous PCR amplifications, thus avoiding the potential contamination of previous PCR products on the current PCR amplification.
This product is particularly suitable for the quantitative or qualitative detection of low abundance or high specificity target genes. For example, highly sensitive detection of low abundance mRNA, lncRNA, small RNA, microbial RNA after reverse transcription, and SNP detection of homologous genes or genes with high similarity that are difficult to distinguish using conventional dye methods.
Principle
The probe method qPCR does not use fluorescent dyes such as SYBR Green to stain PCR products. Instead, it employs a DNA probe labeled with a fluorescent group and a quenching group (quencher) targeting the target sequence to be detected by PCR (the probe binding site is usually located between the two primer binding sites). Normally, the quencher on the probe causes fluorescence quenching due to spatial fluorescence resonance energy transfer (FRET). During the PCR reaction amplification of the target sequence, both primers and probes will anneal to the target gene. With the extension of the primer, the 5'→3' exonuclease activity of the Taq enzyme causes the probe bound to the target sequence to be degraded from the 5' end. After the fluorescent group and quenching group of the probe are cut by the Taq enzyme, the action of the quenching group 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 directly proportional to the number of newly synthesized target fragments, allowing for quantitative detection. The probe is usually a linear DNA specific to the target sequence, with a fluorescent group like FAM or HEX at the 5' end, and a fluorescence quenching group like BHQ1, TAMRA, or MGB at the 3' end.
Features
ROX Dye
This product offers both Low ROX and High ROX, making it broadly compatible with fluorescent quantitative PCR machines that either do not require ROX or require Low ROX or High ROX as a reference dye. The role of ROX is to correct fluorescence fluctuations unrelated to PCR, thereby minimizing inter-well variations. Such variations can be caused by various factors, such as pipetting errors and sample evaporation. Different fluorescent quantitative PCR machines have different requirements for ROX, so please choose either high concentration ROX (High ROX), low concentration ROX (Low ROX), or no ROX Probe based on the specific instrument in use when preparing the reaction system.
Storage
Store in the dark at -20ºC, valid for one year; Store in the dark at 4ºC, valid for one month. Avoid repeated freeze-thaw cycles.
Precautions
Related:
Only for research and not intended for treatment of humans or animals
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