Quantitative Real-time PCR Analysis

The sensitivity of analysis achievable with PCR has led to the technology being adopted across a range of sectors. For many applications a quantitative result is required, which has driven the development of a range of strategies to determine the amount of starting material in a sample. Approaches such as competitive PCR and limiting dilution analysis have been used as routes to quantification, although the variable nature of the PCR process and the amplification of the target to a maximal level irrespective of the starting amount of target limit the accuracy of these methods. The advent of kinetic or real-time PCR has overcome many of the limitations of earlier strategies, by monitoring the increase in product generated during the course of the reaction, in ‘real time’. Quantitative approaches are based on the time or cycle at which amplification is first detected, rather than requiring quantification of PCR products, and the principle is illustrated schematically in Figure 7.1. A range of samples of known target content are usually amplified together with the samples under test, and the accumulation of PCR product in each cycle is determined. Alternatively the signal from two targets may be compared to determine a relative measure of quantification, and this is often used in measurement of gene expression which is considered in more detail in Chapter 9. Here a fluorescent reporter assay is used to monitor increase in fluorescence at each PCR cycle. The point at which the signal becomes detectable, or crosses some arbitrary threshold value, is determined for each standard and sample. These values are then plotted against the amount of target in the standards to produce a calibration curve, and the amount of target in the unknown samples can then be interpolated from the graph.