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:

 

 

Poor Precision

Low sensitivity

Short dynamic range < 2 logs

Low resolution

Non - Automated

Size-based discrimination only

Results are not expressed as numbers

Ethidium bromide for staining is not very quantitative

Post PCR processing
Figure 2: Agarose Gel
As you can see from the figure, the samples in the gel contain 10 copies and 50 copies, respectively. It is hard to differentiate between the 5-fold change on the Agarose gel. Real-Time PCR is able detect a two-fold change (i.e. 10 Vs. 20 copies). 10 copy 50 copy

 

Figure 2: Agarose Gel

As you can see from the figure, the samples in the gel contain 10 copies and 50 copies, respectively. It is hard to differentiate between the 5-fold
change on the Agarose gel. Real-Time PCR is able detect a two-fold change (i.e. 10 Vs. 20 copies).

PCR Phases:
To understand why end-point PCR is limiting, it is important to understand what happens during a PCR reaction.A basic PCR run can be broken up into three phases:

Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.

Linear (High Variability): The reaction components are being consumed, the reaction is slowing, and products are starting to degrade.

Plateau (End-Point: Gel detection for traditional
methods): The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.

Figure 3: PCR Phases

Figure 3 shows three replicates of a sample. The replicates have the same starting quantity. As the PCR reaction progresses, the samples begin to amplify in a very precise manner. Amplification occurs exponentially, that is a doubling of product (amplicon) occurs every cycle.
This type of amplification occurs in the exponential phase. Exponential amplification occurs because all of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.

However, as the reaction progresses, some of the reagents are being consumed as a result of amplification. This depletion will occur at different rates for each replicate. The reactions start to slow down and the PCR product is no longer being doubled at each cycle. This linear amplification can be seen in the linear phase of the reaction. The three samples begin to diverge in their quantities during the linear phase.

 

 

Eventually the reactions begin to slow down and stop all together or plateau. Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is where traditional PCR takes its measurement, also known as end-point detection.
Figure 3 also shows that the three replicate samples, which started out at the same quantity in the beginning of the reaction, reflect different quantities at the plateau phase. Since the samples are replicates they should have identical quantities. Therefore, it will be more precise to take measurements during the exponential phase, where the replicate samples are amplifying exponentially.