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.
