The PCR Blog: The Polymerase Chain Reaction

PCR Quantification

Unlike standard PCR, real-time PCR instruments measure the kinetics of product accumulation in each PCR reaction tube. Generally, no product is detected during the first few PCR cycles as the fluorescent signal is below the detection threshold of the instrument.

Most combinations of machine and fluorescence reporter are capable of detecting the accumulation of amplicons before the end of the exponential amplification phase. During this time the efficiency of PCR is often close to 100% giving a doubling of the quantity of product at each cycle. As product concentrations approach the nanogram per microlitre level the efficiency of amplification falls primarily because the amplicons re-associate during the annealing step. This leads to a phase during which the accumulation of product is approximately linear with a constant level of net synthesis at each cycle. Finally, a plateau is reached when net synthesis approximates zero.

Quantification in real-time PCR is done by measuring the number of cycles required for the fluorescent signal to reach a threshold level or the second derivative maximum of the fluorescence versus cycle curve. This cycle number is proportional to the number of copies of template in the sample. Real-time PCR quantification applications are discussed in detail in Bustin and Nolan (2009) Analysis of mRNA Expression by Real-Time PCR In: Real-Time PCR Logan, Edwards and Saunders, eds.; Wurmbach (2009) Validation of Array DataIn: Real-Time PCR Logan, Edwards and Saunders, eds.; and Wiseman (2009) Real-Time PCR: Application to Food Authenticity and Legislation In: Real-Time PCR Logan, Edwards and Saunders, eds.

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Labels: fluorescence, PCR instruments, PCR machines, quantitation, real-time pcr

PCR Quantification

Real-Time PCR Machines

PCR machines with integrated fluorimeters and a mechanism for transferring excitation light from a source into the reaction vessel and then from the sample to a detector are required for real-time PCR. The heating blocks that are the mainstay of the standard PCR machine present several technical challenges in conversion to application in real-time PCR machines. The main problem being that the light must be channelled through the lid of the block and the cap of the reaction vessel across an air gap and then into the sample. Emitted light must then take the return path. Although blocks are used by several real-time PCR machines including the first commercial real-time PCR machine (Applied Biosystems 7700), the difficulties associated with them have led to the development of alternative designs for real-time pcr machines.

The LightCycler (LC24) was the forerunner of PCR machines that use air as the heating/cooling medium. Thermal transfer via air has the advantage of greater uniformity and rapidity than can be achieved on block-based PCR machines, besides allowing shortening of the light path. As well as differing in the choice of heating medium real-time PCR machines also provide a range of options for the light source and detection of fluorescence. Current, real-time PCR machines tend to allow the excitation and detection of multiple dyes so that internal standards and multiplex reactions are possible. There is also a tendency to build in a bias toward the use of either universal donor or universal recipient chemistry.

The cost of real-time PCR machines has fallen in tandem with continual improvement in their capability and accuracy. This has been the result of competition, the volume of sales and the introduction into the marketplace of improved designs dependent on new technology. These trends are unlikely to be reversed and will contribute to the growth in the popularity of real-time PCR. Real-time PCR machines are described and discussed in more detail in Real-Time PCR Machines (Logan and Edwards 2009. Chapter 2. Real-Time PCR: Current Technology and Applications. Caister Academic Press, Norfolk, UK.

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Labels: fluorimeters, PCR instruments, PCR machines, real-time pcr, thermal cyclers

Real-Time PCR Machines