Here, we report a detailed procedure for the exact quantification of minute amounts of nucleic acids by competitive PCR. This technique entails the co-amplification of a target DNA or cDNA in a biological sample together with a known quantity of a target-specific standard, the competitor, which is added exogenously to the sample and is almost identical to the DNA region to be amplified. Competitive PCR offers the advantage to render the PCR reaction independent of the number of amplification cycles, since any intra-assay variation has the same effect on both target and competitor. Since the final ratio between target and competitor amplification products exactly reflects the initial ratio between the two species, and being the amount of added competitor known, competitive PCR allows the determination of the exact number of molecules of target, with an accuracy that is still unsurpassed by any other alternative procedure, including real-time PCR. The protocols described in this chapter cover most of the possible applications of competitive PCR, including the quantification of an mRNA transcript and the simultaneous determination of multiple targets.

The renaissance of competitive PCR as an accurate tool for precise nucleic acid quantification.

GIACCA, MAURO
2010

Abstract

Here, we report a detailed procedure for the exact quantification of minute amounts of nucleic acids by competitive PCR. This technique entails the co-amplification of a target DNA or cDNA in a biological sample together with a known quantity of a target-specific standard, the competitor, which is added exogenously to the sample and is almost identical to the DNA region to be amplified. Competitive PCR offers the advantage to render the PCR reaction independent of the number of amplification cycles, since any intra-assay variation has the same effect on both target and competitor. Since the final ratio between target and competitor amplification products exactly reflects the initial ratio between the two species, and being the amount of added competitor known, competitive PCR allows the determination of the exact number of molecules of target, with an accuracy that is still unsurpassed by any other alternative procedure, including real-time PCR. The protocols described in this chapter cover most of the possible applications of competitive PCR, including the quantification of an mRNA transcript and the simultaneous determination of multiple targets.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2552520
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