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The iCycler iQ and Amplifluor Detection Systems for Analysis of Genetically Modified Organisms in Foodstuffs, Rev A

fluorescent reporter molecule is used to track the progress of the PCR reaction. The fluorescence of a reaction increases as the amount of amplified DNA increases until a point is reached where the level of fluorescence rises significantly above background. The point at which this occurs is known as the threshold cycle. Since at this point PCR is operating at close to maximum efficiency, the threshold cycle number bears a linear relationship to the amount of target DNA in the reaction. The amount of target DNA in a test sample can be calculated by comparing the samples threshold cycle to a standard curve obtained from the threshold cycles of reference standards.

Many systems exist to introduce the fluorescent reporter molecule to the reaction. The Amplifluor universal amplification and detection system (Intergen) utilizes a molecular energy transfer system that uses an acceptor moiety to quench fluorescence from an excited fluorophore (Nazarenko et al. 1997). The fluorophore (fluorescein) and quencher (DABSYL) are located on the same oligonucleotide primer. The UniPrimer primer has a universal format, as it will bind to any amplified DNA containing a unique recognition sequence that can be added to existing PCR primers. The UniPrimer will only emit fluorescence when incorporated into amplification products; unincorporated UniPrimer does not fluoresce. It is also possible to obtain custom primers with the UniPrimer sequence already included. In this application note, we describe the combined use of the Amplifluor universal amplification and detection systems chemistry and the iCycler iQ real-time PCR detection systems instrumentation for the detection and quantitation GMOs in foodstuffs.



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