Real-time PCR has become a well-established procedure for quantifying levels of gene expression, as well as gene rearrangements, amplifications, deletions or point mutations. Its power resides in the ability to detect the amount of PCR product (amplicon) at every cycle of the PCR, using fluorescence. Several approaches have been employed to detect PCR products. The most popular ones are based on specific binding of hydrolysis of hybridization probes to the target sequence. Another approach uses fluorescent dyes, which bind to the double-stranded PCR product non-specifically. Although both assays are potentially rapid and sensitive, their principles of detection and optimization are different, as is the resulting cost per assay. Here we will explore the development and rigorous testing of a real-time PCR assay using the inexpensive SYBR® Green I technology.

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