The principle of qPCR lies in the continuous monitoring of DNA amplification during the reaction, achieved through the use of fluorescent dyes or probes.
Amplification:
Like conventional PCR, qPCR involves repeated cycles of denaturation, annealing, and extension to amplify a target DNA sequence. If the starting material is RNA, a reverse transcription step is performed first to generate complementary DNA (cDNA).
Fluorescence Detection:
• Intercalating Dyes: Dyes such as SYBR Green bind to double-stranded DNA and emit fluorescence proportional to the amount of DNA generated.
• Probe-Based Detection: Probes like TaqMan contain a fluorescent reporter and quencher, providing target-specific fluorescence upon successful amplification.
Real-Time Quantification:
The fluorescence intensity is measured during each cycle, allowing the accumulation of DNA to be tracked in real time. The cycle at which fluorescence surpasses a set threshold (Ct value) reflects the initial quantity of the target DNA.
Quantification Methods:
qPCR can provide absolute quantification using standard curves, or relative quantification (comparing target expression to reference genes).