- Semi-Quantitative Polymerase Chain Reaction (sqPCR) is a conventional PCR-based technique used to estimate the relative abundance of nucleic acids in a sample.
- Unlike quantitative PCR (qPCR), which measures the accumulation of amplified products in real time, sqPCR relies on endpoint analysis after the PCR amplification process is completed.
- This method assesses the intensity of DNA amplicons following gel electrophoresis and staining, typically using intercalating dyes such as ethidium bromide. The fluorescence intensity of these bands, visualized under UV light, serves as a proxy for the relative amount of the target nucleic acid present in the original sample.
- The sqPCR technique involves amplification of a specific target region using primers and a DNA polymerase in a thermal cycler. After a defined number of cycles, the reaction is stopped, and the PCR products are resolved on an agarose gel. The resulting DNA bands are then compared either visually or by densitometric analysis, often relative to a housekeeping gene or internal control, to account for variability in template input and reaction efficiency. The assumption is that the band intensity correlates with the starting amount of template nucleic acid, enabling semi-quantitative comparisons between samples.
- While sqPCR provides a convenient and cost-effective approach for analyzing gene expression or detecting specific nucleic acids, it has several limitations. Because it analyzes products at the endpoint of the PCR reaction—after amplification has plateaued—the relationship between band intensity and template concentration can be nonlinear and less accurate. Factors such as primer-dimer formation, non-specific amplification, and saturation of the amplification reaction can affect quantification. Moreover, gel electrophoresis and imaging introduce variability in signal intensity, reducing reproducibility and sensitivity, particularly when detecting low-abundance targets.
- Despite its limitations, sqPCR remains a valuable tool in molecular biology for preliminary screening, validation of cloning experiments, or qualitative assessment of gene expression changes under different experimental conditions. Its simplicity and affordability make it accessible to laboratories without advanced real-time PCR instrumentation. However, for experiments requiring precise and reproducible quantification of nucleic acid levels—such as those involved in clinical diagnostics, mutation detection, or viral load monitoring—quantitative PCR (qPCR) is the preferred method due to its superior accuracy and dynamic range.
