The PCR Blog: The Polymerase Chain Reaction: December 2008

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.

Bibliography:

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.

Bibliography:

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

Real-Time PCR Machines

Real-Time PCR Book

Real-Time PCR: Current Technology and Applications
Publisher: Caister Academic Press
Editor: Julie Logan, Kirstin Edwards and Nick Saunders
Publication date: January 2009
ISBN: 978-1-904455-39-4
This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique. This timely and authoritative volume describes the latest PCR platforms, fluorescent chemistries, validation software, data analysis, and internal and external controls and discusses a wide range of RT-PCR applications including: clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping, and much more.
further information

Labels: books, publications, real-time pcr

Real-Time PCR Book

Real-Time PCR Chemistries

There are two general approaches used to obtain a fluorescent signal from the synthesis of product in Real-time PCR. The first depends upon the property of fluorescent dyes such as SYBR Green I to bind to double stranded DNA and undergo a conformational change that results in an increase in their fluorescence. The second approach is to use fluorescent resonance energy transfer (FRET). These methods use a variety of ways to alter the relative spatial arrangement of photon donor and acceptor molecules. These molecules are attached to probes, primers or the PCR product and are usually selected so that amplification of a specific DNA sequence brings about an increase in fluorescence at a particular wavelength.

A major advantage of the real-time PCR instruments and signal transduction systems currently available is that it is possible to characterise the PCR amplicon in situ on the machine. This is done by analysis of the melting temperature and/or probe hybridisation characteristics of the amplicon within the PCR reaction mixture. In the intercalating dye system, the melting temperature of the amplicon can be estimated by measuring the level of fluorescence emitted by the dye as the temperature is increased from below to above the expected melting temperature. The methods that rely upon probe hybridisation to produce a fluorescent signal are generally less liable to produce false positive results than alternative methods such as the use of intercalating dyes to detect net synthesis of double stranded DNA (dsDNA) followed by melting analysis of the product.

Hybridisation, ResonSense and hydrolysis probe systems give fluorescent signals that are only produced when the target sequence is amplified and are unlikely to give false positive results. An additional feature of the hybridisation, ResonSense and related methods is the possibility to measure the temperature at which the probes disassociate from their complementary sequences. This measurement gives a further verification of the specificity of the amplification reaction. An important feature of many of the probe systems is their compatibility with multiplexing due to the availability of fluorophores with resolvable emission spectra.

from N.A. Saunders in Real-Time PCR: Current Technology and Applications

Bibliography:

Labels: fluorescence, fluorescent dyes, hybridisation, multiplexing, PCR instruments, real-time pcr, ResonSense, signal transduction

Real-Time PCR Chemistries

Real-time PCR: Advantages over standard PCR

Standard PCR requires the identification of the amplified fragment(s) by post-PCR analysis, usually by gel electrophoresis. These methods rely on either the size or sequence of the amplicon. Gel electrophoresis, often used to measure the amplicon size, is both inexpensive and simple to implement. However, size analysis has limited specificity since different molecules of approximately the same molecular weight cannot be distinguished. Gel electrophoresis alone is not a sufficient PCR end-point in many instances, including most clinical applications. Characterisation of the product by its sequence is far more reliable and informative and probe hybridisation assays can be used for this purpose. Such methods are time-consuming and care must be taken to ensure that amplicons accidentally released into the laboratory environment do not contaminate the DNA preparation and clean rooms.

Real-time PCR greatly simplifys amplicon recognition by providing the means to monitor the accumulation of specific products continuously during cycling. All current instruments designed for real-time PCR, measure the progress of amplification by monitoring changes in fluorescence within the PCR reaction vessel. Changes in fluorescence can be linked to product accumulation by a variety of methods.

A further advantage of the real-time format is that the analysis can be performed without opening the tube which can then be disposed of without the risk of dissemination of PCR amplicons or other target molecules into the laboratory environment. Although alternative methods for avoiding PCR contamination are available, containment within the PCR vessel is likely to be the most efficient and cost-effective.

A major drawback of standard PCR formats that rely on end-point analysis is that they are not quantitative because the final yield of product is not primarily dependent upon the concentration of the target sequence in the sample. Real-time PCR overcomes this limitation.

from N.A. Saunders in Real-Time PCR: Current Technology and Applications

Bibliography:

Labels: contamination, quantitation, real-time pcr

Real-time PCR: Advantages over standard PCR