Real-time qPCR is a method of adding fluorescent groups to the PCR system, using the accumulation of fluorescent signals, real-time monitoring of the entire PCR process, and quantitative analysis of unknown templates through standard curves. Real-time qPCR has developed rapidly due to its advantages such as easy operation, high sensitivity, and good repeatability. Now it has been widely used in various fields of life science research, such as gene differential expression analysis, SNP detection, allele detection, drug development, clinical diagnosis, genetic modification research, etc.
The probe is an oligonucleotide, and both ends are labeled with a reporter fluorophore and a quencher fluorophore. When intact, the fluorescent signal emitted by the reporter is absorbed by the quencher. When PCR is amplified, Taq The 5'-3' exonuclease activity of the enzyme cleaves and degrades the probe, separates the reporter fluorescent group from the quencher fluorescent group, and releases a fluorescent signal.
SYBR Green is a DNA binding dye that can be incorporated into double-stranded DNA non-specifically. In the free state, it does not emit fluorescence, but once bound to double-stranded DNA, it can emit fluorescence. During the PCR reaction, as the cycle increases, the amount of double-stranded DNA increases, and the corresponding SYBR Green fluorescence value increases, thereby indicating the running process of the PCR reaction。
UNG enzyme anti-pollution：
UNG enzyme can selectively hydrolyze and break uracil glycosidic bonds in double-stranded or single-stranded DNA containing dU. The most important pollutant in PCR experiments is PCR products. To prevent contamination, dUTP is used to replace dTTP in conventional PCR reaction systems. Generate DNA amplification products containing dUTP, add a 50°C heat preservation step before the next PCR, UNG enzyme can degrade the existing U-DNA contaminants in the reaction system, thereby ensuring the specificity and accuracy of the amplification results , And then the UNG enzyme is inactivated at the denaturation temperature.
SNP mutation detection, gene deletion, gene duplication。
Food safety, pathogenic microorganisms (fungi, bacteria, viruses) copy number testing, gene copy number testing, allele testing。
The purpose of relative quantification is to determine the relative proportion of the content of the target gene in two or more samples without knowing their copy number in each sample. It is mainly suitable for the detection of changes in gene expression.
* Double standard curve method。
* Experimental program
* Primer probe sequence
* DNA quality detection (gel electrophoresis, OD value)
* Primer probe specificity verification (gel electrophoresis detection, sequencing)
* Melting curve
* Standard curve
* Original data and related graphs
* Result analysis