Homogeneous Fluorescent Chemistries for Real-Time PCR

The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Resonance Energy Transfer (FRET). In this chapter we describe these methods in detail, outline the principles of each process, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to show how they may be used on various commercially available thermal cyclers. 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In this chapter we discuss the probe technologies or “reporting chemistries.” In order to give the reader a complete understanding of current technology, the principles of real-time analysis will initially be presented outside the context of any specific commercial platform. The number of these is increasing and it is certain that their capabilities on offer will undergo continued improvement and allow new fluorescent approaches to be realised. At this point it is important to highlight to new users the relationship between the choice of probe system and the instrument. These are inextricably linked: the optical specification and the analysis tools on any given platform greatly influence the applicability and utility of different probe systems. Whilst the main factors for the choice of instrument are often driven by throughput requirements and the initial purchase cost, careful consideration of the reporting chemistry for the required application should be made before purchase since the operation of one chemistry or another may be greatly compromised on some platforms. Equally important is the technical support provided from suppliers. This may be limited for non-supported chemistries and so called “open platforms” from manufacturers that do not support any one chemistry. These suppliers may not be able to provide technical advice for the chemistry of choice for the required application. In this chapter we break down probe technology into a number of components to enable the reader to understand how the different assay systems may be used on current and future fluorimetric thermal cyclers. For new users this will facilitate the implementation of various assays on commercial platforms.