Real-Time PCR Quantitation
The ability to monitor the real-time progress of the PCR completely revolutionizes the way one approachesPCR-based quantitation of DNA and RNA. Reactions are characterized by the point in time during cycling whenamplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixednumber of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increasein fluorescence is observed.Figure 2 shows a representative amplification plot and defines the terms used in the quantitation analysis. Anamplification plot is the plot of fluorescence signal versus cycle number. In the initial cycles of PCR, there is littlechange in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence abovethe baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set abovethe baseline. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescencepasses the fixed threshold. As shown by Higuchi et al.2, a plot of the log of initial target copy number for a set ofstandards versus CT is a straight line. Quantitation of the amount of target in unknown samples is accomplishedby measuring CT and using the standard curve to determine starting copy number. The entire process of calculatingCTs, preparing a standard curve, and determining starting copy number for unknowns is performed by the softwareof the 5700 and 7700 systems.
Figure 2. Model of a single amplification plot, showing terms commonly used in real-time quantitative PCR.
Figure 3. Amplification of the human b-action gene in five-fold dilutions of genomic DNA using the GeneAmp® 5700 Sequence Detection System. (a) Amplification plot showing the change in fluorescence of SYBR® Green I dye plotted versus cycle number. (b) Same data showing the log of the change in fluorescence plotted versus cycle number. (c) Standard curve showing CT values plotted versus the log of the initial amount of genomic DNA.
