Real-Time PCR Platforms

Real-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software. In this chapter the main features of each instrument are compared and factors important to weigh up when deciding on a platform are highlighted. History of Real-time PCR Today it is clear that few techniques have had such a powerful impact on biology than the development of the polymerase chain reaction (PCR). More recently the PCR has become even more sophisticated with the introduction of real-time PCR. Initial work by Higuchi and Current PCR http://www.horizonpress.com/pcrbooks ©Horizon Scientific Press colleagues (Higuchi et al., 1992) first demonstrated the simultaneous amplification and detection of specific DNA sequences in real-time by simply adding ethidium bromide (EtBr) to the PCR reaction so that the accumulation of PCR product could be visualised at each cycle. When EtBr is bound to double-stranded DNA and excited by UV light it fluoresces, therefore an increase in fluorescence in such a PCR indicates positive amplification. Soon afterwards they introduced the idea of real-time PCR product quantitation or ‘kinetic PCR’, by continuously measuring the increase in EtBr intensity during amplification with a charge-coupled device camera (Higuchi et al., 1993). By creating amplification plots of fluorescence increase versus the cycle number, they demonstrated that the kinetics of EtBr fluorescence accumulation during thermocycling was directly related to the starting number of DNA copies. Fewer cycles are needed to produce a detectable signal, when a greater number of target molecules are present. Kinetic monitoring also provided a means whereby the efficiency of amplification under different conditions could be determined, providing for the first time insight into the fundamental PCR processes. Therefore, the principle underlying real-time PCR can simply be defined as the monitoring of fluorescent signal from one or more PCRs cycle-by-cycle to completion, where the amount of product produced during the exponential amplification phase can be used to determine the amount of starting material. The approach described above was not ideal since EtBr binds nonspecifically to DNA duplexes and non-specific amplification products, such as primer–dimers, can contribute to the fluorescent signal and result in quantification inaccuracies. Subsequent refinements, the most significant of which was the introduction of fluorogenic probes to monitor product accumulation, added a greater element of specificity to real-time PCR and provided greater quantitative precision and dynamic range than previous methods. These significant advances to the basic PCR technique not surprisingly led to the development of a new generation of PCR platforms and reagents, which allowed simultaneous amplification and quantification of specific nucleic acid sequences cycle-by-cycle. Indeed a few years after Higuchi coined the term ‘kinetic’ or ‘real-time PCR’ the first