Figure 4. Amplification of a segment of the b-actin gene from human genomic DNA. Samples contained 10 ng human genomic DNA (corresponds to 3300 copies of a single copy gene) and were amplified using the components of TaqMan PCR Reagent Kit (PE Biosystems). (a) Amplification plots of 96 replicates. (b) Detail of cycles 20–28. The abscissa is placed at a DRn value of 0.05 to show the threshold used for calculation of CT. The average final DRn value at cycle 40 is 1.03 ± 0.22 (c.v. = 21.4%). The average CT value is 24.64 ± 0.11. A standard deviation of 0.11 for CT corresponds to a c.v. of 7.9% for calculated starting copy number. Experiment performed by Traci Allen, PE Biosystems.
Figure 3a shows amplification, using the 5700 system, of the human b-actin gene in five-fold dilutions of genomicDNA. In this figure, the change in fluorescence of SYBR® Green I dye is plotted versus cycle number. Six replicateswere run for each DNA amount. Figure 3b shows the same data, but with the log of the change in fluorescenceplotted versus cycle number. The 5700 system software calculated the CT (threshold cycle) for each reaction.The CT values are plotted versus the log of the initial amount of genomic DNA to give the standard curve shownin Figure 3c.
These three plots illustrate the basic principles of real-time PCR quantitation. The higher the initial amount ofgenomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value. CT valuesare very reproducible in replicates because the threshold is picked to be in the exponential phase of the PCR. This isshown in Figure 3b where the threshold intersects the amplification plots in the region where there is a linear relationbetween log of the change in fluorescence and cycle number. In the exponential phase, reaction components are notlimiting and replicate reactions exhibit uniform and reproducible results.
