| Criteria | Semi-Quantitative PCR (sqPCR) | Quantitative PCR (qPCR) | Remarks |
| Purpose | Estimates relative abundance of target nucleic acids by comparing band intensities on a gel. | Measures and quantifies the amount of nucleic acids in real-time, often providing absolute or relative quantification. | qPCR provides real-time data and more accurate quantification compared to endpoint-based sqPCR. |
| Quantification Method | Based on the intensity of amplified DNA bands visualized by gel electrophoresis after the PCR reaction is complete. | Measures fluorescence during the exponential phase of PCR to quantify the DNA/RNA in real-time. | sqPCR is endpoint-based and less accurate; qPCR offers real-time precision and sensitivity. |
| Quantitative Accuracy | Low; provides an approximate measurement of product levels; influenced by plateau effects and non-linear amplification. | High; precise quantification due to detection in the exponential phase before amplification plateaus. | qPCR allows for accurate, reproducible measurements; sqPCR lacks precision, especially at low template concentrations. |
| Sensitivity | Lower sensitivity; difficult to detect small changes in gene expression or target concentration. | High sensitivity; detects small differences in gene expression, low copy numbers, or rare mutations. | qPCR is suitable for detecting subtle variations in nucleic acid quantities. |
| Detection | Requires gel electrophoresis and staining (e.g., ethidium bromide) to visualize PCR products. | Fluorescence detection (SYBR Green or sequence-specific probes) is performed in real-time within a closed system. | qPCR minimizes contamination risk by eliminating post-PCR processing steps. |
| Specificity | Moderate; specificity depends on primer design and visualization may detect non-specific products. | High; especially when using sequence-specific probes. SYBR Green qPCR also benefits from melt curve analysis. | qPCR provides better assurance against non-specific amplification. |
| Reproducibility | Lower; variability in gel loading, staining, and imaging affects reproducibility. | High; automated and standardized data acquisition enhances reproducibility across experiments. | qPCR reduces variability by using automated fluorescence measurements. |
| Quantification Type | Provides relative quantification, often expressed as fold changes by comparing to a control (housekeeping gene) or different sample conditions. | Provides absolute quantification (using standard curves or digital methods) or relative quantification (ΔΔCt method). | qPCR offers flexibility depending on the experimental need—relative or absolute quantification. |
| Dynamic Range | Narrow; reliable only within a limited range of template concentrations. | Wide; linear quantification over several orders of magnitude (often 6-8 log units). | qPCR outperforms sqPCR in dynamic range, making it suitable for diverse template amounts. |
| Screening Potential | Suitable for preliminary screening where exact quantification is not critical (e.g., checking gene expression patterns). | Ideal for detailed screening and precise quantification, including diagnostic applications. | sqPCR is often used to identify candidates for further qPCR validation. |
| Applications | Gene expression profiling (relative changes), clone screening, preliminary assessments, and endpoint PCR verification. | Gene expression analysis (absolute/relative), viral load monitoring, mutation detection, diagnostics, and high-throughput screening. | qPCR is widely used in clinical diagnostics and molecular biology research requiring precision. |
| Cost | Low; requires basic thermocycler, agarose gels, and staining agents. | Higher; requires specialized real-time PCR instruments, fluorescent dyes/probes, and software. | sqPCR is cost-effective for small-scale or low-budget labs; qPCR justifies the cost for accuracy and data quality. |
| Time Required | Longer; additional post-PCR steps (gel preparation, running, staining, imaging) are needed. | Faster; real-time data acquisition eliminates the need for post-PCR processing. | qPCR saves time and reduces contamination risk by using closed-tube systems. |
| Multiplexing Capability | Very limited; typically requires separate reactions and gels for different targets. | High; multiplex qPCR enables simultaneous detection of multiple targets in a single reaction using multiple fluorophores. | qPCR significantly enhances throughput with multiplexing capabilities. |
| Risk of Contamination | Higher; open-tube processing for gel electrophoresis increases contamination risk. | Lower; closed-tube system minimizes contamination post-PCR. | qPCR workflows are better suited for sensitive and contamination-prone assays. |
| Instrument Requirements | Basic thermocycler and gel electrophoresis apparatus. | Real-time PCR machine with fluorescence detection capability. | qPCR requires specialized instrumentation but offers integrated and streamlined data analysis. |
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