| Criteria | SYBR Green qPCR | Sequence-Specific Probe-Based qPCR | Remarks |
| Detection Chemistry | Non-specific DNA-binding dye (SYBR Green I) binds to any double-stranded DNA (dsDNA). | Sequence-specific probes (e.g., TaqMan®, Molecular Beacons) hybridize to the target sequence and emit fluorescence upon degradation or conformational change. | SYBR Green detects all dsDNA, including non-specific products; probes offer high specificity. |
| Specificity | Lower specificity; detects both specific amplicons and non-specific products such as primer-dimers. | Higher specificity; only detects the target sequence where the probe hybridizes. | Probe-based qPCR reduces the risk of false positives from non-specific amplification. |
| Assay Design Complexity | Simpler; only requires design of forward and reverse primers. | More complex; requires design and synthesis of specific primers and fluorescently labeled probes. | Probes increase design time and cost but enhance specificity and application range. |
| Multiplexing Capability | Limited; melt curve analysis allows very basic multiplexing if targets have distinct melting temperatures. | High; multiple probes with different fluorophores allow simultaneous detection of multiple targets in one reaction. | Probe-based qPCR is better suited for multiplexing, improving efficiency in multi-target assays. |
| Cost | Lower; requires only primers and SYBR Green dye. | Higher; additional expense for custom-labeled probes and optimized reaction conditions. | SYBR Green is cost-effective for basic research; probe-based assays are justified in diagnostic or complex applications. |
| Sensitivity | High sensitivity but prone to interference from non-specific amplification. | Very high sensitivity with excellent target specificity, even in complex samples. | Probes improve accuracy and sensitivity, particularly for low-abundance targets. |
| Data Validation | Requires post-PCR melt curve analysis to confirm product specificity. | No melt curve typically needed; probe hybridization ensures specificity. | Melt curves are critical for verifying specificity in SYBR Green assays. |
| Applications | Suitable for gene expression analysis, simple target detection, preliminary screening, and educational labs. | Ideal for diagnostic applications, SNP genotyping, mutation detection, pathogen detection, and multiplex assays. | SYBR Green qPCR is frequently used for basic research; probe-based qPCR is standard in diagnostics and translational research. |
| Screening Potential | Good for broad, preliminary screening of gene expression or presence/absence of targets in large sample sets. | Excellent for targeted screening requiring high specificity, such as clinical diagnostics and mutation screening. | SYBR Green is useful for early-phase exploratory research; probes are preferred for confirmatory, high-specificity screening. |
| Risk of Contamination | Moderate; non-specific products and contamination can lead to misinterpretation. | Low; high specificity reduces risk of false positives from contamination. | Probes offer greater assurance in clinical and high-stakes testing. |
| Throughput | Moderate; limited multiplexing capabilities make it less suited for high-throughput applications. | High; supports multiplexing and automation, ideal for high-throughput applications. | Probe-based qPCR is the method of choice for high-throughput diagnostic and screening programs. |
| Instrument Requirements | Standard real-time PCR instruments compatible with SYBR detection. | Requires instruments capable of detecting multiple fluorescence channels (for multiplexing). | Modern qPCR machines typically support both SYBR Green and probe-based assays. |
