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
