The amplification phases can be viewed differently to assess the PCR phases. The figures that follow show the phases of PCR in a Logarithmic scale view and a Linear scale view (Figures 4 and 5). Applied Biosystems Sequence Detection
System Instruments can view the data in both forms.
Problems with detection in the Plateau phase of PCR
The following three figures show the plateau affect on 96 replicates and a fivefold dilution series. As stated earlier, the plateau region is the end-point of the reaction and is representative of the amount of product that you would see on
Agarose Gels. The 96 replicates in the exponential phase are very tight in both the linear and logarithmic views.
In the logarithmic view, Figure 7, the plateau for each reaction seems to occur in the same place, but this is solely due to the log scaling of the plot. Figure 6 shows the same 96 replicates in linear view. The reactions show a clear
separation in the plateau phase; therefore, if the measurements were taken in the plateau phase, quantitation would be affected.
The 5-fold dilution series, seen in Figure 8, seems to plateau at the same place even though the exponential phase clearly shows a difference between the points along the dilution series. This reinforces the fact that if measurements
were taken at the plateau phase, the data would not truly represent the initial amounts of starting target material.
Real-Time chemistry provides fast, precise and accurate results. Real-Time PCR is designed to collect data as the reaction is proceeding, which is more accurate for DNA and RNA quantitation and does not require laborious post PCR
methods.
Quantitation
Theoretically, there is a quantitative relationship between amount of starting target sample and amount of PCR product at any given cycle number.Real-Time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR reaction. Traditional PCR methods use Agarose gels or other post PCR detection methods, which are not as precise. As mentioned earlier, the exponential phase is the optimal point for analyzing data. Real-Time PCR makes quantitation of DNA and RNA easier and more precise than past methods.
The 5‵ Nuclease Assay
5 Nuclease Activity
AmpliTaq Gold® DNA Polymerase has 5‵ exo-nuclease activity. The 5‵exo-nuclease activity of AmpliTaq® Polymerase and FRET (Fluorescent Resonant Energy Transfer) makes it possible to detect PCR amplification in Real-Time. The 5‵ exo-nuclease activity of the enzyme acts upon the surface of the template to remove obstacles downstream of the growing amplicon that may interfere with its’ generation. The 5‵ nuclease assay uses this activity in real time detection.
Figure 9: Taq polymerase activity
Figure 10: 5 ‵Exo-Nuclease Activity of Taq Polymerase:
Here the polymerase is adding bases to a growing chain of DNA.Subsequently, the polymerase is removing DNA that is downstream,impeding its’ capability to synthesize the new strand.
FRET (Fluorescent Resonance Energy Transfer)
FRET or Florescent Resonance Energy Transfer technology is utilized in the 5‵ nuclease assay. The principle is that when a high-energy dye is in close proximity to a low-energy dye, there will be a transfer of energy from high to low, Figure 11.
Figure 11: FRET
The 5‵Nuclease Assay
In the 5‵ nuclease assay, an oligonucleotide called a TaqMan® Probe is added to the PCR reagent master mix. The probe is designed to anneal to a specific sequence of template between the forward and reverse primers.
The probe sits in the path of the enzyme as it starts to copy DNA or cDNA. When the enzyme reaches the annealed probe the 5 exonuclease activity of the enzyme cleaves the probe, Figure 12 through 14.
Figure 12: The 5‵Nuclease Assay
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