qPCR, or quantitative polymerase chain reaction, is a technique that amplifies specific nucleic acid sequences while measuring the amount of product formed in real time using fluorescent signals.
Explanation
Unlike conventional PCR, which only provides endpoint detection, qPCR quantifies DNA or RNA by monitoring fluorescence at each amplification cycle. The reaction contains primers targeting the sequence of interest, a thermostable DNA polymerase and a fluorescent reporter. As the target sequence is replicated, either a dye such as SYBR Green binds to double‑stranded DNA or a sequence‑specific probe (e.g., TaqMan) emits a signal when cleaved by polymerase. The fluorescence increases proportionally with the amount of amplicon, and a threshold cycle (Ct) is calculated when the signal rises above background. By comparing Ct values to a standard curve or reference gene, researchers can determine absolute copy numbers or relative expression levels. When the starting material is RNA, reverse transcription is performed prior to amplification (qRT‑PCR) to assess gene expression. qPCR requires careful optimisation of primer specificity, reaction efficiency and proper controls to ensure reliable quantification.
Applications and Techniques
qPCR is widely used in diagnostics and research. Clinical laboratories quantify viral loads of HIV, hepatitis B and SARS‑CoV‑2 to monitor infection and treatment response. The technique is central to genetic testing for copy number variations and prenatal screening. In molecular biology, qPCR measures gene expression changes in response to stimuli, normalised to housekeeping genes. Environmental microbiologists use qPCR to estimate microbial abundance in water or soil samples. The choice between SYBR Green and probe‑based assays depends on the need for specificity; probes reduce false positives but are more costly. High‑throughput instruments and automated sample preparation have made qPCR an indispensable tool across disciplines.
By combining amplification and quantification in a single step, qPCR delivers rapid, sensitive and accurate measurements of nucleic acids. Its versatility has made it a cornerstone of modern molecular diagnostics and gene expression analysis.
Related Terms: PCR, RT‑PCR, DNA amplification, Ct value, Gene expression