Are SYBR Green I Real-Time PCR assays less specific than TaqMan probe assays?
The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design is poor. TaqMan assays will not generate a signal for any non-specific amplification whereas SYBR Green I assays might. However, non-specific amplification will affect the amplification efficiency and sensitivity of TaqMan assays in the same way as SYBR Green I assays, even though the amplification is not detected. When designing primers for either system, it is important to avoid primer sets that generate any non-specific amplification products. With SYBR Green I assays, the ability to perform melt curve analysis is advantageous for primer design, as any non-specific amplification can be detected and identified in the melt curve. For TaqMan assays, detecting non-specific amplification usually requires another post-PCR analysis method such as agarose gel
