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.