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PrimeTime qPCR Assays

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SYBR® to 5’ Nuclease Assays

Researchers who use intercalating dyes, like SYBR® Green I, for qPCR experiments typically do so for the advantage they bring in lowered price and reduced turnaround time. Custom primers are inexpensive and are received quickly, and intercalating dyes replace the need for an assay-specific modified probe. However, the disadvantages to using these reagents are significant. First, intercalating dyes bind to any double-stranded DNA, not just to the desired amplification product. This means that the signal generated could be from cross-reacting genes, primer-dimers, or other non-specific amplification. Second, effective primers are difficult to properly design and extra melt curves and gels are required to prove specific target amplification.

5’ nuclease assays, such as PrimeTime® qPCR Assays, include the addition of a hydrolysis probe; this assay dependent probe increases specificity by only generating signal when the correct target is amplified. This increase in specificity allows for simpler assay design and a higher success rate. In addition, because the fluorescence is specific to the gene target, you can use probes with different dyes to measure more than one target in a single tube and save precious sample.

Before PrimeTime qPCR Assays, the disadvantages to using 5’ nuclease assays were cost and synthesis turnaround time of the probe; custom probes take longer to receive and are expensive relative to primers. However, with PrimeTime qPCR Assays, these are no longer a problem. PrimeTime qPCR Assays are estimated to be shipped within 2–4 days of order and are affordably priced. In addition, you have the choice of three different synthesis scales to ensure that you are not paying for unused reactions.

2 Comments

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