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What is the difference in specificity when using an intercalating dye (e.g., SYBR Green) vs. a 5’ nuclease assay probe (e.g., PrimeTime® qPCR Assays) to detect PCR product?
Intercalation dyes such as SYBR® Green intercalate into any double-stranded DNA in the sample. They will detect the amplicon of interest but also additional nonspecific products and primer dimers. Thus, use of intercalation dyes can result in a false positive signal and loss of sensitivity when examining products with low copy numbers. Along with the primer set, PrimeTime® qPCR Assays include a probe that is specific to the target product. Therefore, signal will only result when the actual product of interest is amplified and will not result from nonspecific products or primer dimers. This provides greater specificity in the assay.