Though both standard PCR and real-time PCR follow a similar procedure, there are many advantages to real-time PCR. One of the primary advantages of real-time PCR is the ability to identify amplified fragments during the PCR process. Real-time PCR measures the amount of the product during the exponential phase whereas standard PCR measures product during the plateau phase. It is more effective to measure during the exponential phase because measurements taken during the plateau phase do not always clearly indicate the quantity of starting material.
Standard PCR requires post-PCR analysis, possibly agarose gel electrophoresis; it identifies the product either by size or sequence. Though running gel electrophoresis is relatively inexpensive, it is time-consuming and non-automated. It is also low in specificity, since molecules of the same or similar weights cannot be easily differentiated. Gel electrophoresis alone is not suitable for endpoint analysis for most laboratory purposes. The use of probe hybridization is often used for characterization of the product by its sequence. Though this method is more reliable and informative, it is time-consuming and expensive. ELISA detections are also time-consuming. Real-time PCR eliminates these needs. Amplicon recognition is achieved by monitoring the accumulation of specific products during each cycle.
Another advantage of real-time PCR over standard PCR is that the entire process from amplification to analysis is performed in the same tube. This differs from standard PCR where the PCR product is moved and manipulated into other formats. As a result, there is a decreased possibility of contaminating the product with real-time PCR methods.
