qPCR (real-time PCR) protocol explained

qpcr quantitative pcr or real-time pcr is an effective technique to quantitatively determine the presence of a target sequence or gene in a sample up to date qpcr is the gold standard method to test patients for viral infections such as covid19 for that a swap is taken which may contain viral material the rna is extracted and reversed transcribed into dna the dna can then be analyzed with qpcr in a light cycler in conventional pcr a dna sequence of interest is amplified the presence or absence of that dna sequence is detected in an endpoint analysis after reaction

completion during quantitative pcr or real-time pcr the accumulation of the amplification product is visualized with fluorescent reporters and the reaction progression is monitored after each cycle in qpcr the product amplification is therefore observed in a quantitative perspective with each pcr cycle the amount of pcr product doubles which can be detected as an increase in fluorescence this generates a classic qpcr curve a defined signal intensity has to be exceeded until the product is abundant enough so that the fluorescence is detectable over background the cycle number at which enough amplified product has been accumulated to exceed that

threshold is referred to as the ct value qpcr is mainly performed using one of these two essays in the cyber green assay the fluorescent reporter is an intercalating dye that binds to double-stranded dna the tacman qpcr method works with probes specifically designed to bind the sequence of interest upon synthesis of the complementary dna strand the probe is cleaved which results in a fluorescent signal in the cyber green qpcr method one pcr cycle starts with the separation of the two dna strands of the amplicon during denaturation the primers bind to their complementary parts flanking the sequence

of interest the polymerase starts with these synthesis the cyber green dye binds the double-stranded dna fragments and the fluorescence is monitored after each cycle in the tachman assay short dna probes are provided which are attached to a fluorescent reporter at the five prime end the reporter activity is suppressed by the three prime quencher as long as they are in near proximity the strands separate in the denaturation step after this the primers can bind and the checkman probes are designed to specifically bind in between the target sequence of the amplicon the tuck dna polymerase has a

5-prime exonuclease activity which will chew away the target specific probe cleavage of the tagman probe separates the reporter from the quencher which leads to the emission of a fluorescent signal that can be detected after each cycle in both assays the increase of fluorescence along product amplification generates a curve during gene expression analysis multiple genes can be compared the number of cycles after which the signal intensity reaches the threshold is shown as the ct value the presence of gene 1 is detected after 21 cycles whereas the presence of gene 2 is detected in cycle 25. in

general low ct values go along with high dna concentrations and high ct values mean that signals are detected late indicating low dna concentration quantitative pcr finds applications in various fields it can be used for diagnostics to detect infections with pathogens and it is frequently used for gene expression analysis for more scientific content check out the videos on this channel and make sure to subscribe to stay updated thanks