For Reference Only
Description
This tutorial will discuss the evolution of traditional PCR methods towards the use of Real-Time chemistry and instrumentation for accurate quantitation.
Objectives
This tutorial will provide an understanding of the following:
Limitations of traditional PCR
Introduction to Real-Time PCR
Advantages of Real-Time chemistries over traditional PCR methods
The Evolution of PCR to Real-Time
PCR has completely revolutionized the detection of RNA and DNA. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring.
Figure 1: Real-Time PCR Evolution
Real-Time Vs Traditional PCR
Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use Agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction.
Limitations of End-Point PCR
Agarose gel results are obtained from the end point of the reaction. Endpoint detection is very time consuming. Results may not be obtained for days. Results are based on size discrimination, which may not be very precise. As seen later in the section, the end point is variable from sample to sample. While gels may not be able to resolve these variabilities in yield, real-time PCR is sensitive enough to detect these changes. Agarose Gel resolution is very poor, about 10 fold. Real-Time PCR can detect as little as a two-fold change! Some of the problems with End-Point Detection:
